The Role of Melatonin in Affecting Cell Wall Disassembly and Chilling

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Food and Beverage Chemistry/Biochemistry

The Role of Melatonin in Affecting Cell Wall Disassembly and Chilling Tolerance in Cold-Stored Peach Fruit Shifeng Cao, Kun Bian, Liyu Shi, Hsiao-Hang Chung, Wei Chen, and Zhenfeng Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02055 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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Journal of Agricultural and Food Chemistry

1

The Role of Melatonin in Affecting Cell Wall Disassembly

2

and Chilling Tolerance in Cold-Stored Peach Fruit

3 4

Shifeng Cao a, Kun Bian a, Liyu Shi a, Hsiao-Hang Chung b, Wei Chen a,

5

Zhenfeng Yang a, *

6 7 8 9 10

a

11

Ningbo, 315100, People’s Republic of China

College of Biological and Environmental Sciences, Zhejiang Wanli University,

12 13

b

Department of Horticulture, National Ilan University, Yilan, 26047, Taiwan

14 15 16 17

Running Title: Melatonin-Mediated Chilling Tolerance of Peach Fruit

18

* Corresponding author. Tel.: +86-574-88222229. Fax: +86-574-88222991. email:

19

[email protected]

20 21 22 23 24 25 26 1

ACS Paragon Plus Environment


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ABSTRACT: Melatonin reportedly increased chilling tolerance in postharvest peach

28

fruit during cold storage but information on its effect on cell wall disassembly in

29

chilling injured peaches is limited. In this study, we investigated the role of cell wall

30

depolymerization in chilling tolerance induction in melatonin-treated peaches.

31

Treatment with melatonin at 100 µM alleviated chilling symptom (mealiness)

32

characterized by decrease in fruit firmness and increase in juice extractability in

33

treated peaches during storage. Loss of neutral sugars such as arabinose and galactose

34

in both 1,2-cyclohexylenedinitrilotetraacetic acid (CDTA)- and Na2CO3-soluble

35

fractions was observed at 7 d in treated peaches but the contents increased after 28

36

days of storage. Atomic force microscopy (AFM) analysis revealed that the

37

polysaccharides width in CDTA-and Na2CO3-soluble fractions in treated fruit were

38

mainly distributed in a shorter range as compared to the control fruit. In addition,

39

expression profiles of a series of cell wall-related genes showed that melatonin

40

treatment maintained the balance between transcripts of PpPME and PpPG

41

accompanying with up-regulation of several other genes involved in cell wall

42

disassembly. Taken together, our results suggested that the reduced mealiness by

43

melatonin was probably associated with its positive regulation on numerous cell wall

44

modifying enzymes and proteins thus the depolymerization of cell wall

45

polysaccharides in the peaches treated with melatonin was maintained and the treated

46

fruit can soften gradually during cold storage.

47 48 49

KEYWORDS: Melatonin, peach fruit, chilling, mealiness; cell wall; AFM

50 51 52 2


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INTRODUCTION

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Peach (Prunus persica), as a typical temperate fruit, can ripen and deteriorate

55

quickly at ambient temperature. Cold storage is used to slow down these processes.

56

However, cold temperature storage at 2.2-7.6 oC for several weeks can induce chilling

57

injury (CI) called mealiness (woolliness), characterized by a lack of juiciness and a

58

mealy texture in some cultivars of peaches.1,2 It shortens storage life of postharvest

59

peaches and reduces consumer acceptance.1 Therefore, effective method to enhance

60

chilling tolerance has been an urgent demand and further mechanism of physiological

61

response to low-temperature stress of harvested peach fruit should be investigated.3

62

Melatonin (N-acetyl-5-methoxytryptamine), as an effective free-radical scavenger,

63

was discovered in playing an important role in regulating stress response, plant

64

growth, and development.4-9 It has been reported that treatment with melatonin

65

attenuated

66

cold-stress-induced shrinkage and disruption of carrot cell plasma membranes were

67

almost completely alleviated by melatonin treatment.10 Our previous study has

68

revealed that exogenous melatonin treatment increased chilling tolerance and induced

69

defense response in cold-stored peach fruit,

70

understood.

cold-induced

apoptosis

in

carrot

11,12

suspension

cells,

and

the

but the underlying basis is poorly

71

It is well known that several cell wall modifications, such as the solubilisation or

72

depolymerisation of pectin and matrix glycans and loss of neutral sugars from pectin

73

side chains, are associated with normal ripening in postharvest peaches. These

74

changes are of considerable importance in fruit texture.13 Mealiness in cold-stored

75

peaches is related to the abnormal cell wall dismantling during cold storage.14 Cold

76

storage affects numerous cell wall-modifying genes and enzymes, with leading to

77

alteration of cell wall metabolism and further mealiness in peaches subjected to 3



78

chilling. Compared with normal juicy peach fruit, mealy fruit have a decreased

79

solubilization and depolymerization of middle lamella homogalacturonans, and a

80

reduced mobilization of polymeric arabinan from molecules strongly associated with

81

cellulose,14,15 which is caused by the actions of a range of cell wall modifying

82

enzymes and proteins.13,14

83

In the present work, the depolymerization of cell wall polysaccharides was

84

analysed. Meanwhile, the expression of genes that may contribute to their degradation

85

was monitored in order to evaluate the potential contribution of each gene to

86

mealiness alleviation in melatonin treated peach fruit.

87

MATERIALS AND METHODS

88

Melatonin Treatment

89

Peach fruit (Prunus Persica Batsch cv. Hujing) were harvested at commercial

90

maturity from experimental farm of Fenghua Peach Fruit Research Institute (Ningbo,

91

China). The peaches were picked and transported to laboratory quickly and then

92

selected in uniform size and randomly divided into two groups. The first group was

93

immersed into solutions of 100 µM melatonin for 120 min, whereas the second group

94

was soaked in sterile deionized water for 120 min and considered as the control.11,12

95

Afterwards, all fruit were air-dried at room temperature for approximately 30 min,

96

then transferred to 4 oC and 80% relative humidity for 28 d. Fruit samples were taken

97

before melatonin treatment (0 day) and at 7-day intervals during storage for

98

measurements. Each treatment was repeated three times, and the experiment was

99

conducted twice.

100

Texture and Extractable Juice Measurement

4


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Fruit firmness was measured by TPA program with a TMS-Touch Full Touch

102

Properties Analyzer (Federal Trade Commission, U.K.). Extractable juice rate was

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estimated from the weight loss from placental tissue plugs in response to low-speed

104

centrifugation from flesh tissue of 5 fruit. Four plugs (7 mm wide and 10 mm thick)

105

were placed over sterile cotton in a 50 ml centrifuge tube and centrifuged for 10 min

106

at 1700 g at room temperature. The results are expressed as fresh weight loss of the

107

tissue plugs after centrifugation.

108

Preparation for Fractionation of Cell Wall Polysaccharides

109

Cell wall polysaccharides were prepared and fractionated as described by Yoshioka

110

et al with some modification.16 One gram of frozen and homogenized tissues with 4

111

mL ice cold ethanol in a 15 mL centrifuge tube were centrifuged at 5000 × g for 10

112

min. The solution was filtered out and the remaining insoluble cell wall extracts were

113

washed with 5 mL cold 80% (v/v) ethanol, treated with 1 mL Tris-buffered (pH 8.0)

114

phenol for 1 h, and then precipitated with 5 mL of ethanol at -25 oC. Proteins and

115

lipids were removed after treated with 5 mL of chloroform/methanol (1:1, v/v) for 30

116

min at room temperature and washed with 5 mL of acetone. Precipitate was collected

117

by centrifugation at 2000 × g for 10 min. Cell wall polysaccharides obtained above

118

were incubated at room temperature for 12 h with 5 mL of 50 mM

119

1,2-cyclohexanediaminetetraacetic acid (CDTA) which preset to pH 6.0 with 50 mM

120

sodium acetate. CDTA treated solution was gathered for the CDTA-soluble fraction.

121

The precipitate was subsequently incubated with 5 mL of 100 mM Na2CO3 containing

122

0.1% NaBH4 for 6 h at room temperature. Na2CO3 treated solution was gathered for

123

the Na2CO3-soluble extract. Cell wall pectins extracted with CDTA, Na2CO3 solutions

124

were dialyzed against distilled water.

5



125

Determination of Neutral Sugar Composition

126

Dialyzed cell wall materials from CDTA-soluble (CSP) and Na2CO3-soluble (SSP)

127

extracts were freeze-dried, and then lysated by 5 mL of 2 M trifluoroacetic acid in a

128

15 mL digestive tube with nitrogen filled in at 110 oC for 6 h. The neutral sugar

129

hydrolysis was concentrated and determined by gas chromatography (SHIMADZU,

130

Japan) according to the method described.17 The mole proportion of each neutral

131

sugar in total neutral sugars was calculated.

132

Atomic Force Microscopy (AFM) Imaging

133

AFM (NSC15, MikroMasch, Wilsonville, OR, USA) was manipulated at 30-40%

134

relative humidity and around 25℃. Pectin solutions were diluted to 10-30 µg/mL and

135

then agitated at 40 ℃ for 48 h. A small volume (5 µL) of diluted solutions was

136

pipetted onto the mica surface and air dried for 1 h in a dust-free environment before

137

using the AFM image with non-tapping mode at the scan rate of 1.5 Hz.18,19 The scan

138

size of probe was set at 12 × 12 µm, but the images of height mode for analysis was

139

splitted into suitable size areas from original ones and color scale range at 5 or 10 nm

140

to acquire better vision. Several images of different zones were examined and offline

141

analyzed with Nanoscope software (version. 7.20).

142

RNA Extraction and cDNA Synthesis

143

Frozen peach tissues were carefully grounded in liquid nitrogen. Total RNA was

144

extracted using Plant RNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) according

145

to the manufacturer’s instructions. The RNA was treated with amplification grade

146

RNase-free DNase1 (Omega Bio-Tek Inc., Norcross, GA, USA) to remove any DNA

147

contamination prior to cDNA synthesis. Reverse transcription (RT) was carried out 6


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using 2 µg of total RNA and the SuperRT First Strand cDNA Synthesis Kit (CWBIO,

149

Beijing, China) as recommended by the manufacturer.

150

Quantitative Real-Time PCR (qPCR)

151

QPCR reactions were performed with an Mx3000P qPCR System (Agilent

152

Stratagene, Santa Clara, CA, USA) in triplicates using gene specific primers

153

(Supplementary Table S1). Two-step qPCR analysis was performed with SYBR Green

154

PCR master mix (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). The thermal

155

cycling conditions consisted of an initial denaturation at 95 °C for 7 min, and then for

156

40 cycles as follows: denaturation at 95 °C for 15 s combined with each primer

157

specific annealing temperature ranged from 50 °C to 60 °C for 30 s, then completed

158

with a melting curve analysis program.11,12 QPCR data was calibrated relative to

159

PpTEF2 (JQ732180) expression level at zero time for each treatment, following the

160

2−∆∆Ct method for relative quantification.

161

Statistical Analysis

162

Statistical analysis was performed using the SPSS package program version 16.0

163

(SPSS Inc., Chicago, IL). Student's unpaired T test was used to compare the means at

164

P < 0.05.

165

RESULTS

166

Fruit Firmness and Extractable Juice of Peach Fruit

167

No significant changes were observed in fruit firmness during storage in control

168

peaches, however, it declined gradually in fruit treated with melatonin at 100 µM (Fig.

169

1A). Correspondingly, the extractable juice content in the treated fruit increased along 7



170

with the storage time which was significantly higher than that in control fruit (Fig.

171

1B). Thus, peaches subjected to melatonin treatment did not display any chilling

172

symptoms at the end of the storage. In contrast, the fruit without melatonin treatment

173

became dry and had a mealy texture.

174

Neutral Sugar Composition of Cell Wall Fraction

175

The neutral sugar compositions in CSP and SSP for both control and melatonin

176

treated peaches were characterized. The Ara and Gal contents in CSP and SSP in

177

treated peaches declined dramatically after first 7 days of cold storage, which were

178

significantly lower than those in control fruit (Table 1 and 2). However, the contents

179

increased afterwards, and no significant difference was observed between control and

180

treated peaches. There were significantly higher levels of Xyl and Glc but lower Rha

181

content in the CSP of treated peaches as compared to the control peaches (Table 1).

182

Meanwhile, Fuc content in SSP of peaches treated with melatonin was higher than

183

that in control fruit, however lower Man content was observed in the treated fruit

184

(Table 2).

185

AFM Analysis

186

AFM was a powerful tool to observe and characterize the morphology of complex

187

system at molecular level, therefore, it was used to qualitatively and quantitatively

188

analyse the pectin polysaccharides of peach fruit in this study. Samples for

189

determination were CSP and SSP fractions of peaches with or without melatonin

190

treatment stored at 7 d and 28 d. Typical areas of AFM images were selected for

191

further quantitation. Taking picture in Supplementary Figure 1 as example,

192

polysaccharide commonly possesses microstructures like classical long chain (lc),

193

short chain (sc), branch (br), polymer (p) etc. (Fig. S1). In our present study, CDTA 8


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fraction (CSP) contained more branched components in both control and treated

195

peaches as compared to the Na2CO3 fraction (SSP). In both pectin fractions from

196

peaches subjected to melatonin treatment, the distribution of polysaccharides was

197

looser and markedly fewer branches were found at the end of storage than those after

198

7 days (Fig. 2A). However, in the control peaches, more polymers were observed after

199

at the end of storage (Fig. 2A). In order to provide more information about changes in

200

cell wall polysaccharides, the width distributions of CSP and SSP chains were

201

analyzed further. In regardless of the storage time, width of chains in the CSP in

202

control fruit ranged between 20-110 nm, and mainly between 20-70 nm, while in

203

melatonin treated peaches, it was in the range of 20-80 nm and mainly in 20-40 nm

204

(Fig. 2B). No significant changes were observed in the width of SSP chains in treated

205

fruit during storage with the range between 20-70 nm. However, in the peaches

206

without melatonin treatment, it increased from 20-120 nm at 7 d to 50-130 nm at the

207

end of storage (Fig. 2B).

208

Expressions of Cell Wall Related Genes

209

The levels of 15 transcripts encoding proteins involved in cell wall metabolism

210

were investigated. The genes evaluated were the following: expansins 1-3 (PpExp1-3),

211

endo-β-mannanases

212

polygalacturonases 2 (PpPG2), pectate lyases 1 and 2 (PpPL1-2), pectin

213

methylesterase

214

β-xylosidase(PpXyl) and α-arabinofuranosidase (PpAFR1). During storage, the

215

transcripts of the three PpExps, two PpMans, PpPG2 and PpPME1 increased firstly

216

and declined thereafter in the control peaches. However, the melatonin treatment

217

could induce the abundance of PpExps and PpPG2 but inhibit PpPME1 expression at

1

1

and

2

(PpPME1),

(PpMan1-2),

endo-β-1,4-glucanase

β-galactosidases

9


1and

2

(PpEG4),

(PpGal

1-2),


218

day 14 and 21 (Fig. 3). Meanwhile, significantly higher levels of the two PpGals were

219

also observed in the melatonin treated peaches after 21 days of storage (Fig. 3).

220

Additionally, four cell wall related genes such as PpPLs, PpXyl and PpARF1

221

expressed increasingly within storage time in control fruit. The peaches treated with

222

melatonin displayed higher PpXyl and PpARF1 after 14 days of storage but lower

223

PpPLs (Fig. 3). Differently from all the other cell wall related genes analyzed, the

224

expression of PpEG4 in the non-treated peaches decreased gradually during storage,

225

while melatonin treatment up-regulated its expression at day 14 and 21 (Fig. 3).

226

Expressions of PpFLA Gene Family

227

Expression profiles of 12 members in PpFLA gene family of peach fruit were

228

determined. Among them, PpFLA3 and PpFLA7 was not detectable while expressions

229

of others were shown in Fig. 4. Interestingly, all the PpFLAs in control peaches

230

showed a descending expression during storage. No significant difference was

231

observed in transcripts of PpFLA10 between control and melatonin treated peaches,

232

However, the treatment up-regulated the expression of all remaining PpFLAs at the

233

end of storage.

234

DISCUSSION

235

Chilling injury in peach fruit, manifested as a dry, mealy, woolly (lack of juice)

236

texture with no juice, is genetically influenced and triggered by a combination of

237

storage temperature and storage period.1,2 In present study, firmness of cold-stored

238

peach fruit during the whole storage was basically around 20 N, indicating control

239

fruit lacked the capacity to soften normally during cold storage and chilling symptom

240

like mealiness occurred, which was in agreement with previous report.2 Nevertheless,

241

melatonin-treated peaches softened gradually with a constant decline in fruit firmness 10


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and an increase in juice extractability. Fruit firmness and juiciness are important

243

textural components in the case of postharvest peach fruit. Both of the features are

244

largely determined by the modification and structure of cell walls and the extent and

245

strength of adhesion areas between adjacent cells.20,21 Therefore, our results suggested

246

that the application of melatonin could enhance chilling tolerance by regulating

247

alterations of cell wall metabolism in postharvest peaches during cold storage.

248

As the main reason for softening, augment of soluble pectin is caused by many

249

factors. Among of them, the loss of neutral sugars such as Ara or Gal can increase

250

wall porosity and thus allow access of other hydrolase enzymes to their substrate

251

which induces pectin solubilization indirectly.22 Ara and Gal are mainly enriched in

252

rhamnogalacturonan (RG)-Ι side chains, important to the structure and functions of

253

cell walls.23 The progressive loss of large arabinan and galactan side chains during

254

ripening is thus likely to alter cell wall properties (rigidity/flexibility) and intercellular

255

attachments, which may affect pectin solubilization.20,24 This phenomenon happens in

256

majority of fruits and loss of Ara or Gal is the most obvious feature.25,26 Mealiness in

257

peaches and nectarines has been associated with a reduced Gal loss from

258

CDTA-soluble polyuronides, and a loss of Ara from most or all cell wall fractions.27-31

259

Similarly, in our study, losses of Gal and Ara from both CDTA and Na2CO3-soluble

260

extract were absent in mealy peaches, indicating a decreased solubilization and

261

depolymerization of middle lamella homogalacturonans and RG-I from the primary

262

wall in the control peaches subjected to chilling. However, in melatonin treated fruit,

263

the levels of Gal and Ara declined dramatically at the first 7 days of storage,

264

suggesting that an Ara or Gal-containing molecule tightly attached to the wall is

265

metabolized to a more soluble form in juicy fruit.14 In addition, the AFM analysis

266

revealed that width of linear saccharides was generally arranged in a narrower range, 11



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which also indicated that polysaccharides are more likely to be hydrolyzed after

268

melatonin treatment. But it is worth noting that after the dramatic decline of Gal and

269

Ara levels in peaches treated with melatonin, the contents of these two neutral sugars

270

increased significantly at late storage which might be related with the fact that the

271

loosely and tightly bound

272

depolymerization and the released Ara and Gal accumulated at the end of storage.

273

Although no significant difference was observed in the contents of Gal and Ara in

274

control and treated peaches at the end of storage, the CDTA and Na2CO3 fractions in

275

the treated peaches had more cleavage and shorter length of the main backbones

276

demonstrated by AFM analysis. Taken together, our results suggested that under

277

chilling stress, the depolymerization of polymers in the peaches treated with

278

melatonin were maintained in a manner similar to normal ripe fruit and pectins

279

consequently became more easily extractable with enhanced amounts of Ara and Gal

280

at the end storage. Therefore, the treated fruit can ripen to a juicy texture during cold

281

storage. However, in the control peaches, the cell wall polysaccharides were not

282

depolymerized further, the incidence and severity of mealiness symptoms increased

283

progressively.

284

matrix glycans

in treated peaches continued

The development of mealiness in peaches is the result of actions in cell wall

285

modified

enzymes

like

pectin

methylesterase

286

endo-polygalacturonase (PG, EC 3.2.1.15), endo-1,4-β-glucanase (EC 3.2.1.4),

287

β-xylosidase (EC 3.2.1.37), β-galactosidase (EC 3.2.1.23) and arabinosidase (EC

288

3.2.1.55).32 Other non-enzymatic proteins like expansin, also play an important role in

289

mealiness development.33 It has been proposed that relatively high PME and low PG

290

activities in chilling injured fruit leads to an accumulation of de-methylesterified

291

pectins which are not subsequently depolymerized and contributes to the mealiness 12


(PME,

EC

3.1.1.11),

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phenotype in postharvest peaches.14,31,34 In present study, along with the progress of

293

mealiness in control peaches, the transcript abundance of both PpPME1 and PpPG2

294

increased but with a more significant trend in PpPME1 during the first 21 days of

295

storage, which resulted into the imbalance between these two genes and finally

296

mealiness in the cold stored fruit. However, in melatonin treated fruit, lower PpPME1

297

but higher PpPG2 expression was observed, indicating a normal hydrolysis and

298

depolymerization of pectin can be maintained in the treated fruit during cold storage

299

which was associated with the chilling injury inhibition. In addition, our results also

300

showed that melatonin treatment increased the transcripts of PpEG4 encoding

301

endo-1,4-β-glucanase in treated peaches, indicating higher solubilization level of

302

cellulose and hemicelluloses in the treated fruit, which was agreement with previous

303

study that the mealy texture in peaches was associated with a decrease in

304

endo-1,4-β-glucanase activities and its mRNA level.31 A possible role of β-xylosidase,

305

β-galactosidase and arabinosidase in the tolerance to chilling has been previously

306

suggested in peach fruit, as higher transcript levels of PpXyl and higher activities of

307

β-galactosidase and arabinosidase were observed in a tolerant cultivar in comparison

308

to a susceptible one.15 Thus, the higher expression of PpXyl, PpGALs, and PpARF1

309

could be related to the tolerance of peach fruit induced by melatonin treatment. In

310

terms of expansins, Obenland et al reported that the expression of expansin mRNA

311

and protein was strongly suppressed in mealy tissue,35 indicating a possible role for

312

expansin in the development of this disorder. In present study, transcript levels of all

313

three PpExps were higher in melatonin treated peaches, leading to further alteration of

314

cell wall metabolism. Altogether, after comparing the expression of more than 10

315

putative cell wall-related genes between the control and melatonin treated peaches,

316

the reduced mealiness by melatonin was probably associated with its positive 13



317

regulation on numerous cell wall modifying genes thus affect disassembly of cell wall

318

pectins in postharvest peaches.

319

Fasciclin-like arabinogalactan proteins (FLAs) are a distinct subclass of

320

arabinogalactan proteins (AGPs) that, in addition to AGP motifs, are characterized by

321

containing specific one or two fasciclin domains, which functioned in cell

322

adhesion.36 Knockout of FLA4 in Arabidopsis resulted in abnormal cell expansion,

323

thinner cell walls and a reduction in the rays of cellulose across the seed mucilage

324

inner layer.37 Meanwhile, AtFLA11 and AtFLA12 contribute to stem strength by

325

regulating cellulose deposition and to stem elasticity by affecting the integrity of the

326

cell wall matrix.38 FLA1 from cotton is reported to play an important role in

327

modulating the biosynthesis of cell wall polysaccharides during fiber development.39

328

The concomitant expression of FLA genes with xyloglucan remodeling in apple fruit

329

suggested that the proposed cell adhesion and plant mechanical implications of FLA

330

proteins associate specific xyloglucan structures.40 Analysis of the tomato genome

331

sequence revealed that several FLAs showed a substantial decrease in transcript

332

abundance after the mature green stage.41 All of these results suggested that FLAs can

333

have an important role in fruit texture modification as they have been proposed to

334

function in cell wall cross-linking or as pectin plasticizers.42 In present study, among

335

the 9 FLAs detected in peach fruit, the transcripts abundance of FLA genes such as

336

PpFLA1, PpFLA2, PpFLA9 and PpFLA10 declined dramatically with very low

337

expression of other FLA genes in control peaches under chilling stress. However, the

338

melatonin treatment significantly up-regulated most of these FLA genes after 21 or 28

339

days of storage. Consistently, the contents of Ara and Gal started to accumulate

340

rapidly from 21 days. All these indicated that in the melatonin treated peaches, due

341

largely to the higher expression of FLA genes together with other genes such as PpXyl, 14


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PpGals and PpARF1, the Ara and Gal contents at the end of storage could arise from

343

depolymerization or from increased solubility of inherently smaller polymers.

344

In summary, the application of melatonin treatment alleviated mealiness in

345

postharvest peach fruit during cold storage. The treated peaches could soften

346

gradually and display a solubilization and depolymerization of cell wall

347

polysaccharides during cold storage. The expression of cell wall-related and FLA

348

genes was regulated positively by melatonin suggesting that mealiness in cold-stored

349

peaches is coordinated by melatonin at the transcriptional level, and that constitutive

350

transcription involved in cell wall disassembly is required for the chilling induction in

351

melatonin treated peaches.

352

ACKNOWLEDGEMENTSc

353

This study was supported by the National Natural Science Foundation of China

354

(31371866) to Z.Y. and National Natural Science Foundation of China (31571905) to

355

S.C., the Natural Science Foundation of Zhejiang Province (Q15C200013) to W.C.

356

and the Natural Science Foundation of Jiangsu Province (BK20171127) to S.C.

357

ABBREVIATIONS

358

AFM:

atomic

force

microscopy;

AFR:

α-arabinofuranosidase;

CDTA:

359

1,2-cyclohexanediaminetetraacetic acid; CI: chilling injury; CSP: CDTA-soluble

360

pectin;

361

arabinogalactan proteins; Gal: β-galactosidases; Man: endo-β-mannanases; PCR:

362

polymerase chain reaction; PG: polygalacturonases; PL: pectate lyases; PME: pectin

363

methylesterase; q-PCR: quantitative real-time PCR; SSP: Na2CO3-soluble pectin; TPA:

364

texture profile analysis; Xyl: β-xylosidase

EG:

endo-β-1,4-glucanase;

Exp:

expansins;

15


FLA:

fasciclin-like


365

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366

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treatment increases chilling tolerance and induces defense response in harvested

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21



Page 22 of 30

497

Table 1 Neutral sugar content of CDTA-soluble pectin extracts (CSP) of peaches with or without melatonin treatment during cold

498

storage

499

7d

21d

28d

0d

500

CK

Melatonin

CK

Melatonin

CK

Melatonin

501

Ara

43.58 ± 1.27a

30.30 ± 2.82b

5.38 ± 2.30e

28.25 ± 1.04d

30.90 ± 1.81b

29.35 ± 1.96bc

26.48 ± 0.81cd

502

Rha

6.48 ± 0.97e

6.74 ± 0.44e

3.35 ± 0.21f

11.06 ± 0.59c

8.55 ± 0.16d

12.01 ± 0.67b

13.05 ± 0.60a

503

Fuc

10.25 ± 1.24b

14.46 ± 1.11a

10.58 ± 4.50b

11.99 ± 0.30ab

14.40 ± 1.48a

10.36 ± 0.36b

9.71 ± 0.75b

504

Xyl

5.35 ± 0.25cde

7.32 ± 0.67bc

26.37 ± 2.74a

7.99 ± 0.56b

18.92 ± 0.95cd

3.25 ± 0.09e

14.37 ± 0.08de

Gal

23.54 ± 1.56a

13.87 ± 4.08b

5.22 ± 0.86c

16.78 ± 3.80b

17.77 ± 0.71ab

14.94 ± 0.67b

13.74 ± 0.43b

Glc

2.03 ± 0.22c

2.77 ± 0.41c

23.67 ± 2.46a

1.49 ± 0.20c

2.62 ± 0.64c

1.84 ± 0.21c

5.61 ± 1.14b

Man

23.73 ± 0.85e

24.52±0.93de

29.24± 0.85a

25.42 ±0.40cd

21.46 ± 0.36f

26.54 ± 0.23bc

27.01 ± 0.55b

505 506 507 508 509 510

Means in a column followed by a different letter differ significantly at P = 0.05 by Student's unpaired T test. Data are accompanied by standard deviations of the means. Ara: Arabinose; Rha: rhamnose; Fuc: fucose; Xyl: xylose; Gal: galactose; Glc: glucose; Man: mannose.

511 512

22


Page 23 of 30


513

Table 2 Neutral sugar content of Na2CO3-soluble pectin extracts (SSP) of peaches with or without melatonin treatment during cold

514

storage 7d

21d

28d

0d

515 516 517

CK

Melatonin

CK

Melatonin

CK

Melatonin

Ara

53.66 ± 0.58a

32.09 ± 0.62b

17.3 ± 2.39d

28.73 ± 0.08bc

31.38 ± 2.22bc

27.58 ± 1.85bc

26.79 ± 1.71c

Rha

7.32 ± 0.21d

7.35 ± 0.98d

6.79 ± 1.18d

11.05 ± 0.25a

10.32 ± 0.91bc

9.46 ± 0.31cd

10.56 ± 1.11ab

Fuc

13.72± 1.13cd

11.91 ± 0.78d

36.3 ± 0.37a

16.81 ± 0.26c

20.65 ± 1.37b

13.68 ± 0.59cd

13.39 ± 0.54cd

Xyl

2.34 ± 0.13c

5.09 ± 0.58bc

9.41 ± 0.45a

6.23 ± 0.60c

8.71 ± 0.14ab

10.26 ± 0.97a

10.29 ± 0.61a

Gal

12.57 ± 1.56ab

13.36 ± 0.67ab

5.23 ± 0.61c

8.54 ± 0.86bc

11.17 ± 0.92abc

14.84 ± 0.43ab

15.56 ± 0.78a

Glc

4.18 ± 0.72d

5.96 ± 0.61b

5.91 ± 0.43b

8.63 ± 0.66a

3.01 ± 0.57e

4.89 ± 0.12cd

5.19 ± 0.37bc

Man

21.34 ± 0.51c

25.2 ± 0.67a

23.62 ± 0.50b

24.98 ± 0.35a

16.73 ± 0.50e

20.65 ± 0.76d

17.20 ± 0.69e

Means in a column followed by a different letter differ significantly at P = 0.05 by Student's unpaired T test. Data are accompanied by standard deviations of the means. Ara: Arabinose; Rha: rhamnose; Fuc: fucose; Xyl: xylose; Gal: galactose; Glc: glucose; Man: mannose.

23



518

Figure Captions

519

Figure 1. Fruit firmness (A) and extractable juice (B) of control (CK) and melatonin

520

treated fruit during cold storage. Values for firmness are the means ± SE of triplicate

521

samples of five fruit each. Values for extractable juice are the means ± SE of triplicate

522

assays. Asterisks (*) indicate significant differences between CK and melatonin.

523

Figure 2. (A) Atomic force microscopy (AFM)

524

images for CDTA soluble (CSP) and Na2CO3 soluble (SSP) fractions from control and

525

melatonin treated peach fruit at 7 and 28 days of cold storage: (a) image size: 5 × 5

526

µm; (b) image size: 3 × 3 µm; (c) image size: 3 × 3 µm; (d) image size: 3 × 3 µm; (e)

527

image size: 3.5 × 3.5 µm; (f) image size: 3.5 × 3.5 µm; (g) image size: 4.5 × 4.5 µm;

528

(h) image size: 3 × 3 µm; (B) Width of CSP and SSP chains of control and melatonin

529

treated peaches at 7 and 28 days of cold storage. CSP: CDTA soluble fraction, SSP:

530

Na2CO3 soluble fraction.

531

Figure 3. Relative expression level of cell wall related genes in peach fruit treated

532

with or without melatonin during cold storage. All data is presented as a mean of three

533

biological replicates. Transcript abundance was determined using qRT-PCR and was

534

normalized using PpTEF2. PpExp: expansins, PpMan: endo-β-mannanases, PpEG4:

535

endo-β-1,4-glucanase, PpPG2: polygalacturonases 2, PpPL: pectate lyases, PpPME:

536

pectin methylesterase, PpGal: β-galactosidases, PpXyl: β-xylosidase and PpAFR1:

537

α-arabinofuranosidase.

538

Figure 4. Relative expression level of PpFLA gene family of peach fruit treated with

539

or without melatonin during cold storage. All data is presented as a mean of three

540

biological replicates. Transcript abundance was determined using qRT-PCR and was

images and section analysis of AFM

24


Page 24 of 30

Page 25 of 30


541

normalized using PpTEF2.

542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 25



Figure 1. Fruit firmness (A) and extractable juice (B) of control (CK) and melatonin treated fruit during cold storage. Values for firmness are the means ± SE of triplicate samples of five fruit each. Values for extractable juice are the means ± SE of triplicate assays. Asterisks (*) indicate significant differences between CK and melatonin 220x90mm (300 x 300 DPI)


Page 26 of 30

Page 27 of 30


Figure 2. (A) Atomic force microscopy (AFM) images and section analysis of AFM images for CDTA soluble (CSP) and Na2CO3 soluble (SSP) fractions from control and melatonin treated peach fruit at 7 and 28 days of cold storage 378x485mm (300 x 300 DPI)



Figure 3. Relative expression level of cell wall related genes in peach fruit treated with or without melatonin during cold storage. All data is presented as a mean of three biological replicates.Transcript abundance was determined using qRT-PCR and was normalized using PpTEF2. 423x340mm (300 x 300 DPI)


Page 28 of 30

Page 29 of 30


Figure 4. Relative expression level of PpFLA gene family of peach fruit treated with or without melatonin during cold storage. All data is presented as a mean of three biological replicates. Transcript abundance was determined using qRT-PCR and was normalized using PpTEF2. 423x281mm (300 x 300 DPI)



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The Role of Melatonin in Affecting Cell Wall Disassembly and Chilling

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