Interaction between nitric oxide and storage temperature on sphingolipid metabolism of postharvest peach fruit

Journal Pre-proof Interaction between nitric oxide and storage temperature on sphingolipid metabolism of postharvest peach fruit Dandan Huang, Wen Tian, Jianrong Feng, Shuhua Zhu PII:

S0981-9428(20)30119-4

DOI:

https://doi.org/10.1016/j.plaphy.2020.03.012

Reference:

PLAPHY 6091

To appear in:

Plant Physiology and Biochemistry

Received Date: 15 November 2019 Revised Date:

23 February 2020

Accepted Date: 9 March 2020

Please cite this article as: D. Huang, W. Tian, J. Feng, S. Zhu, Interaction between nitric oxide and storage temperature on sphingolipid metabolism of postharvest peach fruit, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2020.03.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Authors contributions Shuhua Zhu and Jianrong Feng conceived the research and edited the final draft. Dandan Huang and Wen Tian performed the research and wrote the original draft. Dandan Huang revised the reviewed manuscript. All authors read and approved the final version of the paper.

1

Interaction between nitric oxide and storage

2

temperature on sphingolipid metabolism of

3

postharvest peach fruit

4

Dandan Huanga,†, Wen Tiana,b,†, Jianrong Fengb,*, Shuhua Zhua,*

5 6 7 8

a

9

Shandong 271018, China

College of Chemistry and Material Science, Shandong Agricultural University, Taian,

10

b

11

Xinjiang 832000, China

Department of Horticulture, College of Agriculture, Shihezi University, Shihezi,

12 13

† They contributed equally.

14 15 16 17

*

Corresponding authors.

18

Shuhua Zhu

19

Jianrong Feng

20

E-mail: [email protected] E-mail: [email protected]

21

Abstract

22

Both nitric oxide (NO) and cold storage have positive effects on the maintenance

23

of fruit quality during storage. However, the roles of NO and storage temperatures in

24

regulating the responses of sphingolipids metabolism to chilling injury of peach fruit

25

during storage remain unknown. Peaches were treated by immersion in distilled water

26

and 15 µmol L-1 NO solution, then stored at 25 °C and 0 °C, respectively. The effects

27

of NO-treatment and storage temperature on the activities of enzymes in sphingolipid

28

metabolism and the contents of sphingolipids in peach fruits were studied. NO

29

maintained higher activities of acid phosphatase (AP) and alkaline phosphatase (ALP)

30

in peach fruits at 25 °C, but promoted the decrease in the activities of AP and ALP at

31

0 °C, suggesting the regulation by NO on AP and ALP could be modulated by

32

temperature. Compared with the storage at 25 °C, cold storage at 0 °C decreased the

33

activities

34

3-ketodihydrosphingosine reductase (KDSR), sphingosine kinase (SPHK), ceramide

35

synthase (CERS), ceramide kinase (CERK), and the contents of sphingosine (SPH),

36

ceramide (CER), sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P),

37

sphingomyelin (SM), and increased the activities of phospholipase C (PLC),

38

phospholipase D (PLD), sphingomyelin synthase (SMS). NO significantly increased

39

the contents of sphingolipid metabolites, and the activities of PLA, KDSR, SPHK,

40

CERS, CERK, but decreased the activities of PLC, PLD, SMS of peaches. The results

41

suggested that NO could maintain sphingolipid metabolism to relieve the response of

42

the postharvest fruit to low temperature.

of

phospholipase

A

(PLA),

alkaline

phosphatase

(ALP),

43

Keywords: sphingolipid, nitric oxide, cold storage, peach, temperature

44

Abbreviation ： AP,

45

ceramide-1-phosphate; CER, ceramide; CERK, ceramide kinase; CERS, ceramide

46

synthase; KDSR, 3-ketodihydrosphingosine reductase; PLA, phospholipase A; PLC,

47

phospholipase C; PLD, phospholipase D; S1P, sphingosine-1-phosphate; SM,

48

sphingomyelin;

49

sphingosine kinase.

50

SMS,

acid

phosphatase;

sphingomyelin

ALP,

synthase;

alkaline

SPH,

phosphatase;

sphingosine;

C1P,

SPHK,

51

1. Introduction

52

Sphingolipid is a lipid that is widely found in eukaryotes and a few prokaryotic

53

biofilms, constituting important structural molecules of cellular membranes; and the

54

balance of relative steady-stats of sphingolipid components plays a significant role in

55

the maintenance of membrane lipid fluidity (Heaver et al., 2018). The signaling and

56

structural effects conferred by each sphingolipid are highly specific, mediate many

57

cellular processes involved in cell cycle arrest, differentiation, migration, aging, and

58

apoptosis in eukaryotes (Duan and Nilsson, 2009; Zheng et al., 2018).

59

Sphingolipid is a complex compound with sphingosine (SPH) as its skeleton

60

(Hannun and Obeid, 2018). Due to the complexity and diversity of the polar head

61

group of sphingolipids, sphingolipids can be classified into ceramide (CER),

62

sphingomyelin (SM) and glycosphingolipids (Lynch and Dunn, 2004). At present,

63

more than 300 sphingolipids have been identified (Kurek et al., 2013). The ab initio

64

synthesis

65

3-ketodihydrosphingosine by serine and palmitoyl-CoA catalyzed by serine

66

palmitoyltransferase (SPT) (Snider et al., 2018). The products of this reaction were

67

subsequently reduced to SPH by 3-ketodihydrosphingosine reductase (KDSR). SPH

68

forms sphingosine-1-phosphate (S1P) by phosphorylation of sphingosine kinase

69

(SPHK) and can be transferred to CER under the action of ceramide synthase (CERS).

70

The sphingolipids biosynthesized in the endoplasmic reticulum and Golgi are

71

transported to the cell membrane to form a membrane lipid bilayer (Cutler et al., 2014;

72

Yamaji and Hanada, 2015). Sphingolipids play an important role in regulating cell

pathway

of

plant

sphingolipids

is

the

production

of

73

senescence and participate in regulating plant response to cold stress (Venable, 2014).

74

Some key enzymes in plant sphingolipid metabolic pathways also affect the metabolic

75

pathways of sphingolipid in plants and regulate the relative homeostasis level of

76

different sphingolipids in plants, thus controlling intracellular signal transduction and

77

other biological processes through these signaling molecules (Wymann and Schneiter,

78

2008; Boini et al., 2017).

79

Nitric oxide (NO) plays multiple roles in plenty of physiological and

80

pathological processes of plants, including programmed cell death, disease resistance,

81

fruit ripening and senescence, and responses to environmental stimulus (Moreau et al.,

82

2008; Wang et al., 2012; Puyaubert et al., 2014; Baudouin and Jeandroz, 2015; Fancy

83

et al., 2017). Recent studies have found that NO and sphingolipid metabolism can

84

interact in plant signal transduction pathways (Perrotta et al., 2008; Guillas et al.,

85

2013). The metabolites of sphingolipids induce the synthesis of endogenous NO in

86

plants, and NO plays a regulatory role in the production and gene expression of

87

sphingolipids in Arabidopsis thaliana (Cantrel et al., 2011). Phospholipase and NO

88

play a synergistic role in regulating plant signal transduction (Gonorazky et al., 2014).

89

Exogenous NO alleviates the chilling injury and regulates the changes in the fatty acid

90

composition of peach fruits during storage (Zhu et al., 2010; Zaharah and Singh,

91

2011; Zhang et al., 2017).

92

As a climacteric fruit, peaches are easy to soften and rot during storage and

93

transportation at ambient temperature (Huan et al., 2018; Wang et al., 2018). Cold

94

storage is a useful method for retarding metabolism and prolonging the storage period

95

of peach fruits. However, peach fruits are sensitive to low temperatures and easily

96

suffer from chilling injury, which manifested as woolliness, browning and losing

97

intrinsic flavor. Thus, chilling injury has become a limit factor in peaches storage and

98

preservation (Cao et al., 2018). As an important factor in coping with cold stress, NO

99

effectively alleviates the chilling injury of peach fruits after harvest (Zhu et al., 2006).

100

Nowadays, the studies on NO regulating the fruit injury due to chilling mainly focus

101

on the effects of NO on fruit storage quality, while the effects of NO on the

102

composition and metabolism of sphingolipid under cold storage conditions are less

103

studied.

104

In this work, the effects of exogenous NO and cold temperature on the activities

105

of sphingolipid metabolism-related enzymes and the contents of sphingolipid

106

metabolites in peach fruits were studied.

107

2. Materials and methods

108

2.1. Fruit materials

109

Peach fruits (Prunus persica (L.) Batsch, cv. Feicheng) were harvested from

110

Feicheng, Taian, Shandong, China. Peaches were randomly selected with uniform in

111

size and no mechanical damage from well-grown plants at a pre-climacteric, but a

112

physiologically mature stage, and then precooled at 0 °C for 24 h. Our previous

113

researches (Jing et al., 2016; Huang et al., 2019) have found that exogenous NO

114

solution at 15 µmol L-1 can exhibit more positive roles in maintaining the quality and

115

prolonging the storage life of peach fruits. Therefore, 15 µmol L-1 NO solution was

116

chosen in this paper. Peach fruits were immersed in 15 µmol L-1 NO solution and

117

distilled water (as control), respectively, for 30 min. After dried with air, the fruits

118

were stored at room temperature (25 °C) and low temperature (0 °C), respectively. At

119

each temperature, there were 3 lots of fruits in each treatment as three replications.

120

Each lot contained 5 cartons with 30 fruits in each carton. Fruits stored at 0 °C were

121

sampled every week and that at 25 °C were sampled every 2 days. Thirty fruits were

122

randomly selected before treatments and expressed as initial samples at day 0 and

123

week 0.

124

2.2 Measurement of firmness

125

The firmness of peaches was measured using a GY-4 durometer (Shanghai

126

Shandu Co., China) equipped with a flat cylindrical probe of 11 mm diameter. Nine

127

peaches were randomly selected from each treatment, and each peach was placed

128

under a probe to record the peak pressure. The results were expressed as N cm-2.

129

2.3 Measurement of soluble solids

130

The soluble solids content (SSC) was measured from a flesh sample with a

131

digital refractometer (Shanghai Cany Precision Instrument Co. Ltd, China). The

132

results were expressed as °Brix.

133

2.4 Measurement of lightness

134

The color was estimated by a CR-10 colorimeter (Konica Minolta, Japan). Nine

135

peaches were randomly selected from each treatment to determine. The results were

136

expressed as lightness (L*).

137

2.5 Measurement of relative electrical conductivity

138

The relative electrical conductivity of peaches was assessed with a DDS-307

139

conductivity meter (Shanghai Yidian Co. Ltd, China). Fifteen slices about 1 mm thick

140

were cut from the same part of nine fruit samples and placed in a small beaker. The

141

initial conductivity of the sample solution was measured after added deionized water

142

to 40 mL, and the initial conductivity was recorded as P0. The conductivity was

143

measured again after 10 minutes and recorded as P1. Finally, the sample solution was

144

boiled for 10 minutes and cooled to room temperature. The conductivity was

145

measured again and recorded as P2. Relative conductivity was calculated using the

146

following formula and expressed as %. Relative conductivity = (P1 - P0)/(P2 -

147

P0)×100 %

148

2.6 Measurement of respiratory rate

149

Peaches fruits (about 1000 g) were placed in a chamber with a volume of about 2

150

L. Then the respiratory rate of peaches fruits was detected by an SY-1022 gas

151

analyzer (Shiya Technology Co. Shijiazhuang, China). Each treatment was repeated

152

three times. The results were expressed as mmol CO2 kg-1 h-1.

153

2.7 Measurement of ethylene production

154

The ethylene production was determined by a gas chromatograph (GC-9A,

155

Shimadzu, Japan) with hydrogen-flame ionization detector according to the method

156

described by Zhu et al., (2006). The detector and gasification chamber temperatures

157

were at 120 °C, column temperature at 70 °C, and the current velocity of N2 and H2

158

were both 40 mL min-1. The rate of ethylene production was expressed as µmol kg-1

159

h-1.

160

2.8 Measurement of the activities of PLA, PLC, and PLD

161

Peach mesocarp (5 g) was homogenized with 5 mL 50 mmol L-1 Tris-HCl buffer

162

(pH 8.0) containing 2 mmol L-1 KCl, 500 mmol L−1 sucrose, 0.5 mmol L−1

163

phenylmethanesulfonyl fluoride (PMSF), 2% (w/v) polyvinylpyrrolidone (PVP). The

164

homogenate was centrifuged at 12,000 ×g for 30 min at 4 °C. The supernatant was

165

collected.

166

The phospholipase A (PLA, EC 3.1.1.4) activity was assayed according to the

167

method of (de Araújo and Radvanyi, 1987). The above 50 µL supernatant was diluted

168

to 500 µL, and then 100 µL diluent was added into 1 mL 5 mmol L−1 sodium

169

phosphate buffer (pH 7.5) containing 3.5 mmol L−1 Lecithin, 7 mmol L−1 Triton

170

X-100, 100 mmol L−1 NaCl, 10 mmol L−1 CaCl2 and 0.35 mmol L−1 neutral red. The

171

absorbance at 522 nm was recorded. One unit of PLA activity was defined as the

172

change of 0.01 in absorbance at A522 in 1 min.

173

The activities of phospholipase C (PLC, EC 3.1.4.3) and phospholipase D (PLD,

174

EC 3.1.4.4) were analyzed according to the procedure described in Mao et al. (2004).

175

The above 0.3 mL supernatant addition to 1mL 0.25 mol L−1 Tris-HCl buffer (pH 7.2)

176

containing 20 mmol L−1 NPPC and 60 % D-sorbitol. The mixture incubated at 37 °C

177

for 1 h, 50 mmol L−1 NaOH was added and then, the absorbance was measured at 400

178

nm. For PLD, the above 0.3 mL supernatant addition to 1mL 50 mmol L−1 Ca–acetate

179

(pH 5.6) containing 27.4 mmol L−1 NPPC, 0.1 mL phosphatase. The mixture

180

incubated at 37 °C for 1 h, 50 mmol L−1 NaOH was added and then, the absorbance

181

was measured at 400 nm. One unit of PLC activity and PLD activity were defined as

182

the change in absorbance of 0.01 at 400 nm per h. These enzymes were shown as U

183

g-1 on a fresh weight basis (FW).

184

2.9 Measurement of the activities of ALP and AP

185

The activities of alkaline phosphatase (ALP, EC 3.1.3.1) and acid phosphatase

186

(AP, EC 3.1.3.2) were determined using Alkaline Phosphatase Assay Kit (Bio Vision,

187

America) and Acid Phosphatase Assay Kit (Bio Vision, America), respectively. The

188

peach mesocarp (5 g) was homogenized with Assay Buffer (at a ratio of 1:10) before

189

3 min of centrifuged at 12,000 ×g at 4 °C. The supernatant was used for ALP and AP

190

activity determinations. Fluorescence intensity was measured at Excitation

191

wavelength (Ex) / Emission wavelength (Em) = 360/440 nm using a fluorescence

192

spectrophotometer (Cary Eclipse, Varian, America), and one unit of ALP and AP

193

activities was defined as the change of 0.1 in fluorescence intensity in 1 sec. These

194

enzymes were described as U g-1 FW.

195

2.10 Measurement of the activities of KDSR, SPHK, and CERS

196

The activity of 3-ketodihydrosphingosine reductase (KDSR, EC 1.1.1.102) was

197

measured according to the method described by (Fornarotto et al., 2006). Peach

198

mesocarp (5 g) were homogenized with 45 mL 10 mmol L−1 potassium phosphate

199

buffer (pH 7.2) containing 250 mmol L−1 sucrose. Homogenate was centrifuged at

200

12,000 ×g at 4 °C for 1 h and the supernatant was collected to determine the activity

201

of KDSR. The absorbance at 340 nm was recorded. One unit of KDSR activity was

202

defined as the change of 0.1 in absorbance at 340 nm per sec and the result was

203

described as U g-1 FW.

204

The activity of sphingosine kinase (SPHK, EC 2.7.1.91) was detected according

205

to the method of (Billich and Ettmayer, 2004). Peach mesocarp (5 g) was

206

homogenized with 10 mL of 10 mmol L−1 potassium phosphate buffer (pH 7.4)

207

containing 1 mmol L−1 dithiothreitol, 1 mmol L−1 ethylenediaminetetraacetic acid, 20 %

208

glycerin, 10 mmol L−1 MgCl2, 1 mmol L−1 Na3VO4, 15 mmol L−1 NaF, 1 mmol L−1

209

PMSF, 20 µmol L−1 ZnCl2, 2 % protease inhibitor, 0.5 mmol L−1 4-dehydropyridoxine.

210

The mixture was centrifuged at 12,000 ×g for 30 min at 4 °C, and the supernatant was

211

then collected. Fluorescence intensity was measured at (Ex) /(Em) = 485/538 nm, and

212

the enzyme activity (1 U) was defined as the change of 0.1 in fluorescence intensity in

213

1 min. The result was expressed as U g-1 FW.

214

The ceramide synthase (CERS, EC 2.3.1.291) activity was measured by a

215

fluorescence spectrophotometer (Cary Eclipse, Varian, America) according to the

216

method of (Kim et al., 2012). Peach mesocarp (5 g) were homogenized in 10 mL 20

217

mmol L−1 HEPES (pH 7.4) containing 25 mmol L−1 KCl, 250 mmol L−1 sucrose, 2

218

mmol L−1 MgCl2, 10 µg mL−1 protease inhibitor. The extract was centrifuged at

219

12,000 ×g at 4 °C for 10 min. The supernatant was then collected as an enzyme

220

extract for the CERS activity assay. The fluorescence intensities at (Ex)/(Em) =

221

485/538 nm were detected, and CERS activity (1 U) was defined as the change of

222

0.01 in fluorescence intensity in 1 h. The data were expressed as U g-1 FW.

223

2.11 Measurement of the activities of CERK and SMS

224

The ceramide kinase (CERK, EC 2.7.1.138) activity in peach fruit was carried

225

out according to (Pettus et al., 2003). Peach mesocarp (5 g) were homogenized with

226

10 mL 20 mmol L−1 HEPES (pH 7.4) containing 50 mmol L−1 NaCl, 1 mmol L−1

227

dithiothreitol, 50 % glycerol, 10 µg mL−1 protease inhibitor. The homogenate was

228

centrifuged at 12,000 ×g for 10 min at 4 °C, and the supernatant was collected for

229

analysis. The fluorescence intensities at (Ex)/(Em) = 485/538 nm were detected, and

230

one unit of CERK activity was defined as the change in fluorescence intensity of 0.01

231

per min. The data were expressed as U g-1 FW.

232

The sphingomyelin synthase (SMS, EC 2.7.8.27) activity was determined

233

according to the method as previously described by (Yeang et al., 2008). The 5 g of

234

peach mesocarp was homogenized with 10 mL 50 mmol L−1 Tris-HCl buffer (pH 7.5)

235

containing 200 mmol L−1 NaCl, 1 mmol L−1 ethylenediaminetetraacetic acid, 2 %

236

protease inhibitor, and then centrifugated at 8,200 ×g, 4 °C for 10 min. The

237

supernatant was used as the crude extract. The fluorescence intensities at (Ex)/(Em) =

238

485/538 nm were detected, and one unit of SMS activity was defined as the change in

239

fluorescence intensity of 0.1 per min. The activity of SMS was expressed as U g-1

240

FW.

241

2.12 Measurement of the contents of sphingolipid metabolites

242

The contents of CER, SPH, SM, S1P, C1P in peach mesocarp were determined

243

with ELISA Kit (Enzyme-linked organism, Shanghai, China). Briefly, the peach

244

mesocarp (5 g) was homogenized with 50 mL 10 mmol L−1 PBS (pH 7.4). The

245

homogenate was centrifuged for 20 min at 3,000 ×g and 4 °C, and the supernatant was

246

collected for measurement of the above indicators. The absorbance at 450 nm was

247

detected. The contents of SPH, C1P, SM, and S1P were expressed as µmol kg-1 FW,

248

and the content of CER was expressed as nmol kg-1 FW.

249

2.13 Statistical analysis

250

Each experiment was designed with three biological replicates. Data are

251

expressed as mean ± standard error (SE). Statistical analysis of the results was

252

performed using a two-way analysis of variance (ANOVA) to evaluate the effects of

253

NO treatment and storage temperature on sphingolipid metabolism. Tukey’s HSD

254

all-pairwise comparisons were used and the probability value (p) of < 0.05 was

255

considered to be statistically significant.

256

3. Results

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3.1 Changes in the physio-chemical parameters of peaches

258

As shown in Fig.1A, the firmness of the peach fruits decreased gradually over

259

time. Exogenous NO delayed the decrease of the firmness of peaches compared with

260

the control both at 25 °C and 0 °C. Compared to storage at 25 °C, cold storage also

261

delayed the decrease of the firmness of peaches. These results indicated that both

262

exogenous NO and cold storage maintained the firmness of peach fruits during

263

storage. However, the maintenance of the firmness by cold storage was more

264

significant than that by NO. Take the data at the third sampling time as an example,

265

the firmness of peaches of the control was 87.77% as higher as that of NO treatment

266

during cold storage, while the firmness of peaches treated by NO stored at 25 °C was

267

55.76% of that stored at 0 °C.

268

The SSC of peaches increased during storage (Fig. 1B). Cold storage

269

significantly delayed the increase in SSC of both control and NO-treated peaches.

270

SSC of control and NO-treated peaches stored at 0 °C were 93.25% and 91.61%,

271

respectively, of that stored at 25 °C at the first sampling time.NO-treated peaches also

272

exhibited lower SSC of peaches compared with control during the whole storage. SSC

273

of NO-treated peaches stored at 0 °C and 25 °C was 93.32% and 95.00%, respectively,

274

as high as that of control peaches at the first sampling time.

275

The L* value of peaches decreased gradually during the cold storage, however, it

276

maintained stably after the second sampling time at 25 °C (Fig. 1C). The L* of

277

NO-treated peaches was higher than that of the control during the whole storage at

278

25 °C. Similar changes were also observed in peaches stored at 0 °C. Compared with

279

storage at 25 °C, cold storage at 0 °C inhibited the decrease in L* of peaches although

280

the L* also decreased during cold storage. And the L* value of control peaches stored

281

at 0 °C was 1.05 times that of 25 °C at the second sampling time. However, the effect

282

of NO on L* of peaches at 25 °C was more significant than that at 0 °C.

283

The significant difference in the relative electrical conductivity in peaches was

284

observed between the NO treatment and the control at both 25 °C and 0 °C (Fig. 1D).

285

The relative electrical conductivity of NO-treated peaches was significantly lower

286

than that of the control during storage. For instance, at the third sampling time, the

287

relative electrical conductivity of NO-treated peaches was 77.47% and 83.97% of that

288

of the control at 25 °C and 0 °C, respectively. The relative electrical conductivity in

289

peaches stored at 0 °C was lower than that of peaches at 25 °C. The relative electrical

290

conductivity of the control and NO-treated peaches during cold storage at the third

291

sampling time was 74.86% and 81.15%, respectively, of that of peaches stored at

292

25 °C. These results showed that peaches treated with NO and stored at 0 °C

293

maintained the lowest relative electrical conductivity during storage.

294

At 25 °C, the respiratory rate of both the control and NO-treated peaches reached

295

a peak at day 4 (Fig. 1E). The respiratory rate of NO-treated peaches was significantly

296

lower than that of the control. The respiratory rate of NO-treated peaches was 84.38%,

297

83.95%, 90.97% of that of the control at day 4, 6, 8, respectively, at 25 °C. At 0 °C,

298

the respiratory rate of the control and NO-treated peaches reached a peak at week 2

299

and 3, respectively. And the respiratory rate of the control was 1.24 times as high as

300

that of peaches treated with NO at week 4 during cold storage. NO could significantly

301

reduce the respiration rate and delay the peak of the respiration rate in peach fruits

302

both at 25 °C and 0 °C.

303

At 25 °C, the ethylene production gradually increased and peaked at day 6, and

304

then decreased (Fig. 1F). The ethylene production of peaches treated with NO was

305

significantly lower than that of control, which was 92.23%, 90.24%, 92.36% of that of

306

the control at day 4, 6, 8, respectively. At 0 °C, the ethylene production in peaches

307

increased during the first 3 weeks and then decreased gradually as the cold storage

308

period extended. The ethylene production of NO-treated peaches was also lower than

309

that of the control at 0 °C. The ethylene production of NO-treated peaches was

310

85.00%, 89.35%, 87.76% of that of the control at week 2, 3, 4, respectively. The

311

ethylene production was effectively inhibited by NO during storage both at 25 °C and

312

0 °C.

313

3.2 Changes in the activities of PLA, PLC, and PLD

314

There was a significant effect of both NO treatment and storage temperature on

315

the activities of PLA, PLC, PLD, and no statistical interaction between the two

316

factors.

317

At 25 °C, NO-treated peaches had significantly higher activity of PLA than the

318

control during the entire storage period, indicating that NO could significantly

319

improve the activity of PLA (Fig. 2A). PLA activity of NO-treated peaches on day 2

320

reached the maximum, which was 1.72 times that of the control. Cold storage

321

decreased PLA activity of peaches. The PLA activity of peaches treated with NO was

322

significantly higher than that of the control at 0 °C and even higher than that of the

323

control at 25 °C, but lower than that of peaches treated with NO at 25 °C.

324

Compared with the storage at 25 °C, cold storage at 0 °C significantly increased

325

PLC activity of peaches during storage (Fig. 2B). NO significantly decreased PLC

326

activity of peaches both at 25 °C and 0 °C. The PLC activity of peaches treated with

327

NO was 58.08%, 68.08%, 88.10%, 64.65% of that of the control at day 2, 4, 6, 8,

328

respectively, at 25 °C. The PLC activity of peaches treated with NO was 53.26%,

329

80.56%, 87.16% of that of the control at week 1, 2, 3, respectively, at 0 °C.

330

PLD activity of the control peaches at 0 °C maintained higher than that of the

331

control at 25 °C (Fig. 2C). Especially after day 4, PLD activity of the control peaches

332

sharply decreased at 25 °C, while that of the control at 0 °C maintained stable from

333

week 2 to 4. At 25 °C, PLD activity of NO-treated peaches was 54.39%, 61.21%,

334

59.49%, 84.96% of the control at day 2, 4, 6, 8, respectively. At 0 °C, PLD activity of

335

NO-treated peaches was 64.02%, 63.10%, 76.95%, 81.91% of that of the control at

336

week 1, 2, 3, 4, respectively.

337

3.3 Changes in AP and ALP activities

338

AP activity of peaches decreased during storage at 25 °C (Fig. 3A). NO

339

significantly increased AP activity of peaches during storage at 25 °C. The AP

340

activities of NO-treated peaches were 1.21, 1.16, 1.45, 1.29 times that of the control at

341

day 2, 4, 6, 8, respectively, at 25 °C. Conversely, NO significantly decreased AP

342

activity at 0 °C. The AP activities of NO-treated peaches during cold storage was

343

78.10%, 79.95%, 77.04%, 76.59% of that of the control at week 1, 2, 3, 4,

344

respectively. There was a significant statistical interaction between NO treatment and

345

storage temperature (two-way ANOVA: F=27.009, p=0.001) on reducing AP activity.

346

The AP activities of NO-treated peaches stored at 0 °C were 52.02%, 64.08%,

347

63.89%, 70.76% of that of stored at 25 °C at the first, second, third, fourth sampling

348

times, respectively.

349

Similar changes were also found in the ALP activity of peaches during storage

350

(Fig. 3B). The statistical interaction between NO treatment and storage temperature

351

on reducing ALP activity was significant (two-way ANOVA: F=11.843, p=0.009).

352

The ALP activities of NO-treated peaches stored at 0 °C were 25.71%, 39.27%,

353

23.24%, 37.00% of that of stored at 25 °C at the first, second, third, fourth sampling

354

times, respectively. Compared with the storage at 25 °C, cold storage decreased ALP

355

activity during storage. NO significantly decreased ALP activity at 0 °C. The AP

356

activities of NO-treated peaches during cold storage was 53.76%, 70.23%, 50.17%,

357

61.78% of that of the control at week 1, 2, 3, 4, respectively.

358

3.4 Changes in the activities of KDSR, SPHK, CERS, CERK, and SMS

359

The activities of KDSR, SPHK, CERS, CERK, and SMS were significantly

360

affected by both NO treatment and storage temperature with all the probability values

361

< 0.05 (two-way ANOVA). However, the statistical interactions between NO

362

treatment and storage temperature were not significant.

363

Cold storage decreased the KDSR activity of peaches but NO increased KDSR

364

activity during storage (Fig. 4A). The KDSR activities of NO-treated peaches was

365

1.38, 1.37, 1.21, 1.43 times that of the control at day 2, 4, 6, 8, respectively, at 25 °C,

366

and was 1.20, 1.74, 1.50, 1.50 times that of the control at week 1, 2, 3, 4, respectively,

367

at 0 °C.

368

Similar effects of cold storage and NO treatment on the activities of SPHK and

369

CERS were also found in peaches (Fig. 4B, 4C). The SPHK activities of NO-treated

370

peaches was 1.41, 1.10, 1.16, 1.15 times that of the control at day 2, 4, 6, 8 at 25 °C,

371

ant was 1.23, 1.21, 1.18, 1.18 times that of the control at week 1, 2, 3, 4 at 0 °C. The

372

CERS activities of NO-treated peaches was 1.34, 1.30, 1.17, 1.11 times that of the

373

control at day 2, 4, 6, 8 at 25 °C, and was 1.16, 1.36, 1.58, 1.12 times that of the

374

control at week 1, 2, 3, 4 at 0 °C.

375

At 25 °C, CERK activity in NO-treated peaches gradually increased from 0.443

376

U g-1 and reached its maximum on day 6, and then decreased to 0.932 U g-1 on day 8

377

(Fig. 4D). Cold storage decreased CERK activities of both the control and NO-treated

378

peaches during storage. And NO significantly improved CERK activity of peaches

379

during storage both at 25 °C and 0 °C. The CERK activities of NO-treated peaches

380

was 1.35, 1.88, 2.67, 2.09 times that of the control at day 2, 4, 6, 8 at 25 °C, and was

381

1.68, 2.49, 2.49, 2.15 times that of the control at week 1, 2, 3, 4 at 0 °C.

382

Compared with the storage at 25 °C, cold storage increased SMS activities of

383

peaches during storage (Fig. 4E). However, NO significantly inhibited SMS activity

384

of peaches during storage both at 25 °C and 0 °C. The SMS activity of NO-treated

385

peaches was only 84.33%, 31.10%, 54.04%, 55.66% of that of the control at day 2, 4,

386

6, 8, respectively, at 25 °C, and was 70.31%, 48.69%, 55.57%, 34.06% of that of the

387

control at week 1, 2, 3, 4, respectively, at 0 °C. These results indicated that peaches

388

treated with NO and stored at 25 °C maintained the lower SMS activity during

389

storage.

390

3.5 Changes in the contents of SPH, CER, S1P, C1P, and SM

391

The contents of SPH, CER, S1P, C1P, and SM were significantly dependent on

392

both NO treatment and storage temperature (two-way ANOVA for all: p<0.05).

393

Further, the statistical interactions between the two factors were not significant

394

(two-way ANOVA for all: p>0.05).

395

Cold storage reduced the contents of SPH, CER, and S1P (Fig. 5A, 5B, and 5C).

396

Compared with the control, the contents of SPH, CER, and S1P of the NO-treated

397

peaches were significantly increased during storage at both 25 °C and 0 °C. The SPH

398

content of NO-treated peaches was 1.13, 1.30, 1.18, 1.21 times that of the control at

399

day 2, 4, 6, 8, respectively, at 25 °C, and was 1.13, 1.15, 1.22, 1.26 times that of the

400

control at week 1, 2, 3, 4, respectively, at 0 °C. The CER content of NO-treated

401

peaches was 1.20, 1.43, 1.24, 1.14 times that of the control at day 2, 4, 6, 8,

402

respectively, at 25 °C, and was 1.29, 1.40, 1.14, 1.25 times that of the control at week

403

1, 2, 3, 4, respectively, at 0 °C. The S1P content of NO-treated peaches was 1.12, 1.03,

404

1.18, 1.19 times that of the control at day 2, 4, 6, 8, respectively, at 25 °C, and was

405

1.07, 1.16, 1.12, 1.20 times that of the control at week 1, 2, 3, 4, respectively, at 0 °C.

406

Both the content of C1P and SM increased in the first period of storage and then

407

decreased at the end of storage (Fig. 5D and 5E). Cold storage also decreased the

408

contents of C1P and SM of peaches, and NO increased the contents of C1P and SM of

409

peaches during storage at both 25 °C and 0 °C. The C1P content of NO-treated

410

peaches was 1.20, 1.20, 1.18, 1.12 times that of the control at day 2, 4, 6, 8,

411

respectively, at 25 °C, and was 1.12, 1.29, 1.22, 1.30 times that of the control at week

412

1, 2, 3, 4, respectively, at 0 °C. The SM content of NO-treated peaches was 1.18, 1.15,

413

1.12, 1.28 times that of the control at day 2, 4, 6, 8, respectively, at 25 °C, and was

414

1.60, 1.36, 1.14, 1.37 times that of the control at week 1, 2, 3, 4, respectively, at 0 °C.

415

Peaches treated with NO and stored at 25 °C maintained higher content of C1P and

416

SM during storage than other treatments.

417

4. Discussion

418

As a common method to prolong the postharvest life of fruit, cold storage is used

419

popularly (Liu et al., 2019). Compared with the storage at 25 °C, cold storage at 0 °C

420

inhibited the respiration rate and ethylene production and maintained the storage

421

quality of peaches. Similar results confirm that storage at 0 °C prolongs the storage

422

life of peach fruit (Liu et al., 2019). Cold storage reduced the activities of PLA, AP,

423

ALP, KDSR, SPHK, CERS, CERK, and the contents of SPH, CER, S1P, C1P, SM,

424

but increased the activities of PLC, PLD, SMS of peaches. As a bioactive molecule,

425

NO exhibited protective effects on the quality of peaches during storage both at 25 °C

426

and 0 °C. The protection by NO on the ripening and the storage quality of fruit has

427

also been found in peach (Huang et al., 2019), sweet pepper (Gonzalez-Gordo et al.,

428

2019), orange (Ghorbani et al., 2017), apple (Chen et al., 2019), table grapes (Zhang

429

et al., 2019), and so on. Ethylene production is an important factor promoting the

430

ripening and senescence of peach fruit and cause the loss of fruit quality during

431

storage. NO inhibited ethylene production in peach fruit both at 25 °C and 0 °C. NO

432

inhibits the activity of 1-aminocyclopropane-1-carboxylic acid oxidase (Zhu et al.,

433

2006), decreases and delays the maximum of ethylene production (Zhu et al., 2010;

434

Zaharah and Singh, 2011), which delays fruit softening and retards color development

435

of peach fruit during storage.

436

Sphingolipids are the structural components of the plasma membrane and other

437

endomembrane systems and also act as signaling molecules in plant response to biotic

438

and abiotic stresses (Xin et al., 2015; Ali et al., 2018; Huby et al., 2019). Sphingolipid

439

content in olive-fruit protoplasts increases at the onset of ripening and reaches a

440

maximum at the onset of ripening and then decreases during fruit ripening (Ines et al.,

441

2018). Sphingolipids are also affected by low temperature, and the sphingolipid

442

signaling in plant response to low temperature is well summered by (Ali et al., 2018).

443

It is reported that both phytosphingosine phosphate (PHS-P) and ceramide phosphate

444

(Cer-P) are specifically biosynthesized in Arabidopsis upon cold exposure (Cantrel et

445

al., 2011; Guillas et al., 2011). However, cold storage decreased the contents of SPH,

446

CER, S1P, C1P, and SM in peaches in this work. The opposite result might be due to

447

the difference between the growing Arabidopsis plants and the postharvest peach fruit.

448

Sphingolipid metabolism is strikingly different between different organs in plants

449

(Luttgeharm et al., 2015). As a growing plant, Arabidopsis can get what it needs from

450

the environment. However, during storage, the postharvest peach fruit is an

451

independent individual, and cannot get support from the plants. So, sphingolipid

452

metabolism in the postharvest peach fruit response to the cold storage might be

453

different.

454

Nitric oxide participates in cold-responsive phosphosphingolipid formation

455

(Cantrel et al., 2011). However, the interplay between NO and sphingolipids is still

456

controversial (Ali et al., 2018). NO modifies the cold-triggered synthesis of PHS-P

457

and Cer-P, but does not affect the cold-responsive formation of phosphatidic acid

458

(PtdOH) in Arabidopsis, so phosphosphingolipid metabolism is regarded as a novel

459

downstream element of NO signaling (Cantrel et al., 2011; Guillas et al., 2011).

460

To reveal the possible interrelationship of the effects of NO treatment and cold

461

storage on sphingolipid metabolism in peach fruit, information concerning the

462

changes of enzyme activities and metabolites is visually represented in Figure 6. As

463

shown in Fig. 6B, NO increased the activities of KDSR, CERS, and CERK, which led

464

to the high contents of sphingosine (SPH), ceramide (CER) and ceramide-1-phosphate

465

(C1P) in peaches during cold storage at 0 °C. With high activities of SPHK, peach

466

fruit treated with NO maintained high content of sphingosine-1-phosphate (S1P). By

467

inhibiting SMS activity, NO reduced the conversion from ceramide to sphingomyelin.

468

Nevertheless, the contents of sphingomyelin (SM) of NO-treated peaches were much

469

higher than that of the control.

470

On the other hand, cold storage up-regulated the activities of SMS, PLD, and

471

PLC, but down-regulated the activities of other enzymes in peach fruit. However, NO

472

down-regulated the activities of SMS, PLD, and PLC, but up-regulated the activities

473

of other enzymes in peach fruit during cold storage. These results suggested that there

474

might be antagonism between NO and cold storage on the sphingolipid metabolism in

475

the postharvest peach fruit. Cold storage is also abiotic stress for peach fruit, and NO

476

could maintain sphingolipid metabolism to alleviate the response of the postharvest

477

fruit to low temperature. And it was interesting that NO decreased the activities of AP

478

and ALP of peaches at 0 °C, which was the same as cold storage do, but NO

479

promoted them at 25 °C. The results of two-way ANOVA also showed there was a

480

significant statistical interaction between NO treatment and the storage temperature. It

481

is suggesting that the roles of NO on the activities of AP and ALP depended on the

482

temperature.

483

The relationship between NO and sphingolipid is still ambiguous. It is reported

484

that sphingolipid metabolism is strikingly different between pollen and leaf in

485

Arabidopsis (Luttgeharm et al., 2015). The difference in sphingolipid metabolism

486

between different organs in the plants also aggravated the complex relationship

487

between NO and sphingolipid metabolism. The physio-biochemical process of the

488

postharvest fruit is different from the plant. NO evolves in prolonging the storage life

489

and maintaining the quality of fruit (Huang et al., 2019; Palma et al., 2019).

490

Sphingolipids also play important roles in response to low temperature (Yan et al.,

491

2019). These preliminary results indicated the effects of NO on sphingolipid

492

metabolism of peaches at different storage temperatures. Storage at low temperatures

493

is popularly used to prolong the life of fruit. However, cold storage easily causes

494

chilling injury. Further works should be done to explore the interplay between NO

495

and sphingolipids in the postharvest fruit during cold storage.

496

Authors contributions

497

Shuhua Zhu and Jianrong Feng conceived the research and edited the final draft.

498

Dandan Huang and Wen Tian performed the research and wrote the original draft.

499

Dandan Huang revised the reviewed manuscript. All authors read and approved the

500

final version of the paper.

501

Conflict of interest statement

502 503 504 505 506

The authors confirm that this article content has no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31470686, 31770724).

507

References

508

Ali U, Li H, Wang X, Guo L, 2018. Emerging roles of sphingolipid signaling in plant response to

509

biotic

510

https://doi.org/10.1016/j.molp.2018.10.001.

and

abiotic

stresses.

Mol

Plant.

11,

1328-1343.

511

Baudouin E, Jeandroz S (2015) Nitric oxide as a mediator of cold stress response: a

512

transcriptional point of view. In: Khan M., Mobin M., Mohammad F., Corpas F. (eds)

513

Nitric Oxide Action in Abiotic Stress Responses in Plants. Springer, Cham. pp 129-139.

514

https://doi.org/10.1007/978-3-319-17804-2_8.

515 516 517

Billich A, Ettmayer P, 2004. Fluorescence-based assay of sphingosine kinases. Anal Biochem 326, 114-119. https://doi.org/10.1016/j.ab.2003.11.018. Boini KM, Xia M, Koka S, Gehr TWB, Li PP-L, 2017. Sphingolipids in obesity and related

518

complications.

519

https://doi.org/10.2741/4474.

Frontiers

in

bioscience

(Landmark

edition)

22,

96-116.

520

Cantrel C, Vazquez T, Puyaubert J, Reze N, Lesch M, Kaiser WM, Dutilleul C, Guillas I,

521

Zachowski A, Baudouin E, 2011. Nitric oxide participates in cold-responsive

522

phosphosphingolipid formation and gene expression in Arabidopsis thaliana. New

523

Phytol 189, 415-427. https://doi.org/10.1111/j.1469-8137.2010.03500.x.

524

Cao S, Shao J, Shi L, Xu L, Shen Z, Chen W, Yang Z, 2018. Melatonin increases chilling

525

tolerance in postharvest peach fruit by alleviating oxidative damage. Sci Rep 8,

526

806-816. https://doi.org/10.1038/s41598-018-19363-5.

527

Chen Y, Ge Y, Zhao J, Wei M, Li C, Hou J, Cheng Y, Chen J, 2019. Postharvest sodium

528

nitroprusside treatment maintains storage quality of apple fruit by regulating sucrose

529

metabolism.

530

https://doi.org/10.1016/j.postharvbio.2019.04.024.

Postharvest

Biology

and

Technology

154,

115-120.

531

Cutler RG, Thompson KW, Camandola S, Mack KT, Mattson MP, 2014. Sphingolipid

532

metabolism regulates development and lifespan in Caenorhabditis elegans. Mech

533

Ageing Dev 143143-144, 9-18. https://doi.org/10.1016/j.mad.2014.11.002.

534

de Araújo AL, Radvanyi F, 1987. Determination of phospholipase A2 activity by a colorimetric

535

assay

536

https://doi.org/10.1016/0041-0101(87)90136-X.

537

using

a

pH

indicator.

inflammation

539

https://doi.org/10.1016/j.plipres.2008.04.003.

541

25,

1181-1188.

Duan RD, Nilsson A, 2009. Metabolism of sphingolipids in the gut and its relation to

538

540

Toxicon

and

cancer

development.

Prog

Lipid

Res

48,

62-72.

Fancy NN, Bahlmann AK, Loake GJ, 2017. Nitric oxide function in plant abiotic stress. Plant Cell Environ 40, 462-472. https://doi.org/10.1111/pce.12707.

542

Fornarotto M, Xiao L, Hou Y, Koch KA, Chang E, O'Malley RM, Black TA, Cable MB, Walker

543

SS, 2006. Sphingolipid biosynthesis in pathogenic fungi: identification and

544

characterization of the 3-ketosphinganine reductase activity of Candida albicans and

545

Aspergillus

546

https://doi.org/10.1016/j.bbalip.2005.11.013.

fumigatus.

Biochim

Biophys

Acta

1761,

52-63.

547

Ghorbani B, Pakkish Z, Khezri M, 2017. Nitric oxide increases antioxidant enzyme activity and

548

reduces chilling injury in orange fruit during storage. New Zealand Journal of Crop and

549

Horticultural Science 46, 101-116. https://doi.org/10.1080/01140671.2017.1345764.

550

Gonorazky G, Distéfano AM, García García-Mata C, Lamattina L, Laxalt AM, 2014. Phospholipases in

551

nitric oxide-mediated plant signaling. Phospholipases in Plant Signaling 20, 135-158.

552

https://doi.org/10.1007/978-3-642-42011-5_8.

553

GonzalezGonzalez-Gordo S, Bautista R, Claros MG, Canas A, Palma JM, Corpas FJ, 2019. Nitric

554

oxide-dependent regulation of sweet pepper fruit ripening. J Exp Bot 70, 4557-4570.

555

https://doi.org/10.1093/jxb/erz136.

556

Guillas I, Puyaubert J, Baudouin E, 2013. Nitric oxide-sphingolipid interplays in plant signalling:

557

a

558

https://doi.org/10.3389/fpls.2013.00341.

new

enigma

from

the

Sphinx?

Front

Plant

Sci

4,

341-347.

559

Guillas I, Zachowski A, Baudouin E, 2011. A matter of fat: interaction between nitric oxide and

560

sphingolipid signaling in plant cold response. Plant Signal Behav 6, 140-142.

561

https://doi.org/10.4161/psb.6.1.14280.

562 563 564 565

Hannun YA, Obeid LM, 2018. Sphingolipids and their metabolism in physiology and disease. Nat Rev Mol Cell Biol 19, 175-191. https://doi.org/10.1038/nrm.2017.107. Heaver SL, Johnson EL, Ley RE, 2018. Sphingolipids in host-microbial interactions. Curr Opin Microbiol 43, 92-99. https://doi.org/10.1016/j.mib.2017.12.011.

566

Huan C, An X, Yu M, Jiang L, Ma R, Tu M, Yu Z, 2018. Effect of combined heat and 1-MCP

567

treatment on the quality and antioxidant level of peach fruit during storage.

568

Postharvest

569

https://doi.org/10.1016/j.postharvbio.2018.07.013.

Biology

and

Technology

145,

193-202.

570

Huang D, Hu S, Zhu S, Feng J, 2019. Regulation by nitric oxide on mitochondrial permeability

571

transition of peaches during storage. Plant Physiol Biochem 138, 17-25.

572

https://doi.org/10.1016/j.plaphy.2019.02.020.

573

Dhondt--Cordelier S, 2019. Sphingolipids: Huby E, Napier JA, Baillieul F, Michaelson LV, Dhondt

574

towards an integrated view of metabolism during the plant stress response. New

575

Phytol https://doi.org/10.1111/nph.15997.

576

Ines C, ParraParra-Lobato MC, Paredes MA, Labrador J, Gallardo M, SaucedoSaucedo-Garcia M,

577

GavilanesGavilanes-Ruiz M, GomezGomez-Jimenez MC, 2018. Sphingolipid Distribution, Content and

578

Gene Expression during Olive-Fruit Development and Ripening. Front Plant Sci 9,

579

28-41. https://doi.org/10.3389/fpls.2018.00028.

580

Jing G, Zhou J, Zhu S, 2016. Effects of nitric oxide on mitochondrial oxidative defence in

581

postharvest

582

https://doi.org/10.1002/jsfa.7310.

583 584

peach

fruits.

J

Sci

Food

Agric

96,

1997-2003.

Kim HJ, Qiao Q, Toop HD, HD, Morris JC, Don AS, 2012. A fluorescent assay for ceramide synthase activity. J Lipid Res 53, 1701-1707. https://doi.org/10.1194/jlr.D025627.

585

Kurek K, Lukaszuk B, Piotrowska DM, Wiesiolek P, Chabowska AM, ZendzianZendzian-Piotrowska M,

586

2013. Metabolism, physiological role, and clinical implications of sphingolipids in

587

gastrointestinal

588

https://doi.org/10.1155/2013/908907.

tract.

Biomed

Res

Int

2013,

908907.

589

Liu H, Jiang W, Cao J, Li Y, 2019. Effect of chilling temperatures on physiological properties,

590

phenolic metabolism and antioxidant level accompanying pulp browning of peach

591

during

592

https://doi.org/10.1016/j.scienta.2019.05.037.

cold

storage.

Scientia

Horticulturae

255,

175-182.

593

Luttgeharm KD, Kimberlin AN, Cahoon RE, Cerny RL, Napier JA, Markham JE, Cahoon EB,

594

2015. Sphingolipid metabolism is strikingly different between pollen and leaf in

595

Arabidopsis

596

Phytochemistry 115, 121-129. https://doi.org/10.1016/j.phytochem.2015.02.019.

as

revealed

by

compositional

and

gene

expression

profiling.

597

Lynch DV, Dunn TM, 2004. An introduction to plant sphingolipids and a review of recent

598

advances in understanding their metabolism and function. New Phytologist 161,

599

677-702. https://doi.org/10.1111/j.1469-8137.2004.00992.x.

600

Mao L, Karakurt Y, Huber DJ, 2004. Incidence of water-soaking and phospholipid catabolism

601

in ripe watermelon (Citrullus lanatus) fruit: induction by ethylene and prophylactic

602

effects of 1-methylcyclopropene. Postharvest Biology and Technology 33, 1-9.

603

https://doi.org/10.1016/j.postharvbio.2003.12.007.

604

Moreau M, Lee GI, Wang Y, Crane BR, Klessig DF, 2008. AtNOS/AtNOA1 is a functional

605

Arabidopsis thaliana cGTPase and not a nitric-oxide synthase. J Biol Chem 283,

606

32957-32967. https://doi.org/10.1074/jbc.M804838200.

607

Palma JM, Freschi L, RodriguezRodriguez-Ruiz M, GonzalezGonzalez-Gordo Gordo S, Corpas FJ, 2019. Nitric oxide in

608

the

609

https://doi.org/10.1093/jxb/erz350.

physiology

and

quality

of

fleshy

fruits.

J

Exp

Bot

70,

4405-4417.

610

Perrotta C, De Palma C, Clementi E, 2008. Nitric oxide and sphingolipids: mechanisms of

611

interaction and role in cellular pathophysiology. Biol Chem 389, 1391-1397.

612

https://doi.org/10.1515/BC.2008.155.

613

Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA, Chalfant CE, 2003. Ceramide

614

kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release. J

615

Biol Chem 278, 38206-38213. https://doi.org/10.1074/jbc.M304816200.

616

Puyaubert J, Fares A, Reze N, Peltier JB, Baudouin E, 2014. Identification of endogenously

617

S-nitrosylated proteins in Arabidopsis plantlets: effect of cold stress on cysteine

618

nitrosylation

619

https://doi.org/10.1016/j.plantsci.2013.10.014.

level.

Plant

Sci

215215-216,

150-156.

620

Snider JM, Snider AJ, Obeid LM, Luberto C, Hannun YA, 2018. Probing de novo sphingolipid

621

metabolism in mammalian cells utilizing mass spectrometry. J Lipid Res 59,

622

1046-1057. https://doi.org/10.1194/jlr.D081646.

623

Venable ME, 2014. Elevation of ceramide in senescence: role of sphingolipid metabolism, In:

624

MA Hayat, (Eds.), Tumor Dormancy, Quiescence, and Senescence, Volume 2: Aging,

625

Cancer, and Noncancer Pathologies. Springer Netherlands, Dordrecht, pp 81-88.

626

https://doi.org/10.1007/978-94-007-7726-2_9

627

Wang Q, Liang X, Dong Y, Xu L, Zhang X, Hou J, Fan Z, 2012. Effects of exogenous nitric

628

oxide on cadmium toxicity, element contents and antioxidative system in perennial

629

ryegrass.

630

https://doi.org/10.1007/s10725-012-9742-y.

Plant

Growth

Regulation

69,

11-20.

631

Wang X, Fu X, Chen M, Huan L, Liu W, Qi Y, Gao Y, Xiao Xiao W, Chen X, Li L, Gao D, 2018.

632

Ultraviolet B irradiation influences the fruit quality and sucrose metabolism of peach

633

(Prunus persica L.). Environmental and Experimental Botany 153, 286-301.

634

https://doi.org/10.1016/j.envexpbot.2018.04.015.

635 636

Wymann MP, Schneiter Schneiter R, 2008. Lipid signalling in disease. Nature Reviews Molecular Cell Biology 9, 162-176. https://doi.org/10.1038/nrm2335.

637

Xin XF, Nomura K, Ding X, Chen X, Wang K, Aung K, Uribe F, Rosa B, Yao J, Chen J, He SY,

638

2015. Pseudomonas syringae effector avirulence protein E localizes to the host

639

plasma membrane and down-regulates the expression of the NONRACE-SPECIFIC

640

DISEASE

641

antibacterial

642

https://doi.org/10.1104/pp.15.00547.

643

RESISTANCE1/HARPIN-INDUCED1-LIKE13 immunity

in

Arabidopsis.

Plant

gene

Physiol

required 169,

for

793-802.

Yamaji T, Hanada K, 2015. Sphingolipid metabolism and interorganellar transport: localization

644

of sphingolipid

645

https://doi.org/10.1111/tra.12239.

enzymes

and

lipid

transfer

proteins. Traffic

16, 101-122.

646

Yan J, Luo Z, Ban Z, Lu H, Li D, Yang D, Aghdam MS, Li L, 2019. The effect of the

647

layer-by-layer (LBL) edible coating on strawberry quality and metabolites during

648

storage.

649

https://doi.org/10.1016/j.postharvbio.2018.09.002.

Postharvest

Biology

and

Technology

147,

29-38.

650

Yeang C, Varshney Varshney S, Wang R, Zhang Y, Ye D, Jiang XC, 2008. The domain responsible for

651

sphingomyelin synthase (SMS) activity. Biochim Biophys Acta 1781, 610-617.

652

https://doi.org/10.1016/j.bbalip.2008.07.002.

653

Zaharah SS, Singh Z, 2011. Postharvest nitric oxide fumigation alleviates chilling injury, delays

654

fruit ripening and maintains quality in cold-stored ‘Kensington Pride’ mango.

655

Postharvest

656

https://doi.org/10.1016/j.postharvbio.2011.01.011.

Biology

and

Technology

60,

202-210.

657

Zhang T, Che F, Zhang H, Pan Y, Xu M, Ban Q, Han Y, Rao J, 2017. Effect of nitric oxide

658

treatment on chilling injury, antioxidant enzymes and expression of the CmCBF1 and

659

CmCBF3 genes in cold-stored Hami melon (Cucumis melo L.) fruit. Postharvest

660

Biology

and

Technology

127,

88-98.

661

https://doi.org/10.1016/j.postharvbio.2017.01.005.

662

Zhang Z, Xu J, Chen Y, Wei J, Wu B, 2019. Nitric oxide treatment maintains postharvest

663

quality of table grapes by mitigation of oxidative damage. Postharvest Biology and

664

Technology 152, 9-18. https://doi.org/10.1016/j.postharvbio.2019.01.015.

665

Zheng P, Wu JJ-X, Sahu SK, Zeng HH-Y, Huang LL-Q, Liu Z, Xiao S, Yao N, 2018. Loss of

666

alkaline ceramidase inhibits autophagy in Arabidopsis and plays an important role

667

during environmental stress response. Plant, Cell & Environment 41, 837-849.

668

https://doi.org/10.1111/pce.13148.

669

Zhu S, Liu M, Zhou J, 2006. Inhibition by nitric oxide of ethylene biosynthesis and

670

lipoxygenase activity in peach fruit during storage. Postharvest Biology and

671

Technology 42, 41-48. https://doi.org/10.1016/j.postharvbio.2006.05.004

672

Zhu S, Sun L, Zhou J, 2010. Effects of different nitric oxide application on quality of kiwifruit

673

during 20 °C storage. International Journal of Food Science & Technology 45,

674

245-251. https://doi.org/10.1111/j.1365-2621.2009.02127.x.

675

Figure Captions 676

Fig.1 Effect of NO treatments on the firmness (A), soluble solids content (B),

677

lightness (C), relative conductivity (D), respiration rate (E) and ethylene production

678

(F) of peach fruit at different storage temperatures. Values represent the mean ±

679

standard error (SE), n = 3 separate experiments. Values with different letters within

680

the same sampling time are significantly different (p < 0.05).

681 682

Fig.2 Effect of NO treatments on the activities of PLA (A), PLC (B) and PLD (C) of

683

peach fruit at different storage temperatures. Values represent the mean ± standard

684

error (SE), n = 3 separate experiments. Values with different letters within the same

685

sampling time are significantly different (p < 0.05).

686 687

Fig.3 Effect of NO treatments on the activities of AP (A) and ALP (B) of peach fruit

688

at different storage temperatures. Values represent the mean ± standard error (SE), n =

689

3 separate experiments. Values with different letters within the same sampling time

690

are significantly different (p < 0.05).

691 692

Fig.4 Effect of NO treatments on the activities of KDSR (A), SPHK (B), CERS (C),

693

CERK (D) and SMS (E) of peach fruit at different storage temperatures. Values

694

represent the mean ± standard error (SE), n = 3 separate experiments. Values with

695

different letters within the same sampling time are significantly different (p < 0.05).

696 697

Fig.5 Effect of NO treatments on the contents of SPH (A), CER (B), S1P (C), C1P (D)

698

and SM (E) of peach fruit at different storage temperatures. Values represent the mean

699

± standard error (SE), n = 3 separate experiments. Values with different letters within

700

the same sampling time are significantly different (p < 0.05).

701

702

Fig.6 (A) Sphingolipid metabolism pathways in peach fruit. (B) Venn diagram

703

showing the interrelationship of the effects of NO treatment and cold storage on

704

sphingolipid metabolism in peach fruit. The sets with various fill colors represent up-

705

or down-regulation of enzyme activities and metabolites by NO treatment or cold

706

storage.

Highlights

NO can significantly delay the release of ethylene and maintain the storage quality of peach fruit. NO significantly increased the contents of SPH, CER and S1P under 25 °C and 0 °C. Cold storage at 0 °C can decreased the activities of 3KSR, SPHK, CERS, CERK, SPH, CER, S1P, C1P and SM. The regulation by NO on AP and ALP could be modulated by temperature.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: