Changes in Cuticular Wax Composition of Two Blueberry Cultivars

45 mins ago - New type of protein interaction observed. Biochemists have long thought that biomolecules must have or develop well-defined structures t...
0 downloads 8 Views 890KB Size
Subscriber access provided by MT ROYAL COLLEGE

Article

Changes in Cuticular Wax Composition of Two Blueberry Cultivars During Fruit Ripening and Postharvest Cold Storage Wenjing Chu, Haiyan Gao, Hangjun Chen, Wei-Jie Wu, and Xiangjun Fang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05020 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Changes in Cuticular Wax Composition of Two Blueberry Cultivars During

2

Fruit Ripening and Postharvest Cold Storage

3 4

WENJING CHU,† HAIYAN GAO,†,* HANGJUN CHEN,† WEIJIE WU,† AND XIANGJUN FANG†

5 6



7

of Post-Harvest Handling of Fruits, Ministry of Agriculture; Key Laboratory of Fruits

8

and Vegetables Postharvest and Processing Technology Research of Zhejiang

9

Province, 298 Middle Desheng Road, Hangzhou 310021, China

Food Science Institute, Zhejiang Academy of Agricultural Science; Key Laboratory

10 11 12 13

*To whom correspondence should be addressed. Telephone: (+86) 571-86406661;

14

Fax (+86) 571-86404378; E-mail: [email protected]

15 16 17

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

18

ABSTRACT: Cuticular wax plays an important role for the quality of blueberry fruits.

19

In this study, the cuticular wax composition of two blueberry cultivars, ‘Legacy’

20

(Vaccinium corymbosum) and ‘Brightwell’ (V. ashei), were examined during fruit

21

ripening and postharvest cold storage. The results showed that wax was gradually

22

deposited on the epidermis of blueberry fruits, and the content of major wax

23

compounds, except that for diketones, increased significantly during fruit ripening.

24

The total wax content was 2-fold greater in ‘Brightwell’ blueberries than that in

25

‘Legacy’ blueberries during fruit ripening. The total wax content of both cultivars

26

decreased during 30 d of storage at 4 °C, and the variation of cuticular wax

27

composition was cultivar-dependent. The content of diketones decreased significantly

28

in ‘Legacy’ blueberries, while the content of triterpenoids and aliphatic compounds

29

showed different fold changes in ‘Brightwell’ blueberries after 30 d of storage at 4 °C.

30

Overall, our study provided a quantitative and qualitative overview of cuticular wax

31

compounds of blueberry fruits during ripening and postharvest cold storage.

32 33

KEYWORDS: Cuticular wax; blueberry; fruit ripening; cold storage; gas

34

chromatography-mass spectrometry

35

2

ACS Paragon Plus Environment

Page 2 of 32

Page 3 of 32

Journal of Agricultural and Food Chemistry

36

INTRODUCTION

37

Blueberry fruits contain high concentrations of bioactive components, such as

38

procyanidins, anthocyanins, flavonols and chlorogenic acid (1-3). These components

39

have antimicrobial, antioxidant, anti-cancer, antidiabetic and cardio-protective

40

activities. Blueberry is a popular fruit worldwide for their high nutritional value and

41

health benefits. However, they are prone to postharvest decay, physical damage,

42

softening, water loss and shriveling. Cuticular wax is a hydrophobic coat which

43

consists of cutin and wax and surrounds the fruit epidermis. It creates an effective

44

barrier against water loss, ultraviolet radiation and microbial invasion (4,5), and plays

45

an important role in the postharvest quality of fruits. Previous studies in sweet cherry

46

have shown that the postharvest quality and storability characteristics of fruits, such

47

as weight loss, firmness and susceptibility to physiological disorders, are greatly

48

affected by the chemical composition, structure and properties of the cuticle (6,7).

49

Mukhtar and others (8) also reported that water loss and shelf life is negatively

50

correlated with the wax content in European plum. Moggia and others (9) found that

51

softening rates in blueberryare highly relevant to the content of ursolic acid in

52

cuticular wax at harvest.

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

53

Cuticular wax is a conspicuous white layer which is formed during fruit ontogeny

54

in a wide range of fruits such as blueberry, grapes and plum (10). The biosynthesis of

55

cuticular wax begins with C16 and C18 fatty acid synthesis in the plastids of

56

epidermal cells. These molecules are then transported to the cytoplasm and further

57

elongated to very-long-chain (VLC) fatty acids by the fatty acid elongase complex in

58

the endoplasmic reticulum. The chain lengths of the VLC fatty acids range from C20

59

to C34. They are converted either into corresponding alkanes, aldehydes, secondary

60

alcohols and ketones or into primary alcohols and wax esters (11,12). The wax load

61

and chemical composition vary widely among different species, cultivars and tissues,

62

and are affected by ripening stages and environmental conditions (13–15). Liu and

63

others (11) compared two cultivars of Citrus sinensis, ‘Newhall’ and ‘Glossy

64

Newhall’, and found that their wax loads varied considerably during fruit ripening.

65

Wu and others (12) observed significant differences in the chemical composition of

66

cuticular wax in three Asian pear cultivars, ‘Kuerle’, ‘Xuehua’ and ‘Yuluxian’, during

67

storage. Pensec and others (16) reported that high levels of triterpenoids existed in the

68

cuticular wax of young grapes, but gradually decreased with a slight increase of

69

neutral triterpenoids during fruit ripening.

4

ACS Paragon Plus Environment

Page 4 of 32

Page 5 of 32

Journal of Agricultural and Food Chemistry

70

In our previous studies, we determined the chemical composition and morphology

71

of cuticular wax in the mature fruit of nine blueberry cultivars (17) and investigated

72

the effects of cuticular wax on the concentrations of bioactive compounds, activity of

73

antioxidant enzymes and other quality indices during cold storage (18). Moggia and

74

others (9) also explored the triterpenoid fraction of cuticular wax and its effect on the

75

postharvest fruit behavior in blueberries. However, only limited information is

76

available regarding the compositional changes in cuticular wax during fruit ripening

77

and cold storage, and thus, the underlying mechanisms for this remain unclear.

78

Therefore, the objective of this study was to assess the chemical composition of

79

cuticular wax in two most widely cultivated highbush and rabbiteye blueberry

80

cultivars in China, ‘Legacy’ (Vaccinium corymbosum) and ‘Brightwell’ (V. ashei),

81

during ripening and cold storage. The results from this study are important in

82

understanding changes in the development and chemical composition of cuticular wax,

83

and its effect on the quality of blueberry fruits.

84 85

MATERIALS AND METHODS

86

Blueberry Samples. Blueberry fruits of two cultivars, ‘Legacy’ (V.corymbosum) and

87

‘Brightwell’ (V.ashei), were respectively hand-picked at three different phenological

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

88

stages (T1, green fruit; T2, red fruit; and T3, dark blue fruit) from a local orchard in

89

Zhejiang Province, China in June 2015. Blueberry fruits were harvested using

90

polyvinyl chloride gloves, placed in plastic baskets, and transported to the laboratory

91

by a refrigerated boxcar (10 °C) within 2 h after harvest. Fruits which were of

92

uniform size and color and showed no signs of physical damage were used for

93

analysis. Fruits at three mature stages (T1, T2 and T3) were analyzed immediately

94

after harvest for examining changes of wax content during fruit ripening. In addition,

95

fruits at mature stage T3 were used of the storage study, and were subpackaged and

96

stored at 4 °C and 90% relative humidity for 30 d.

97

Chemicals and Standards. All solvents and reagents used for analysis and extraction

98

were of analytical grade. Authentic standards of β-amyrin, α-amyrin and lupeol were

99

purchased from Sigma-Aldrich (St. Louis, MO, USA). Oleanolic acid, ursolic acid

100

and n-tetracosane were purchased from J&K Scientific (Beijing, China).

101

Determination of Fruit Surface Area. The fruit surface area was determined as

102

described by Chu et al. (17). The equatorial and polar diameters of each fruit were

103

measured using a digital Verniercaliper (Guanglu Measuring Instrument, Guilin,

104

China), and the fruit surface area was calculated according to the equation shown

105

below:

6

ACS Paragon Plus Environment

Page 6 of 32

Page 7 of 32

Journal of Agricultural and Food Chemistry

106

Surface area = πd2,

107

Where d is the average of the equatorial and polar diameters of each fruit.

108

Extraction and

109

chromatography-mass spectrometry (GC-MS) analysis were performed as described

110

by Chu et al. (17) with minor modifications. In brief, 20 fruits were submerged in 20

111

ml chloroform and gently shaken for 1 min at 25 °C. The extraction was performed in

112

duplicate and the two extracts were combined and filtered, 100µL (1µg/µL)

113

n-tetracosane, an internal standard, was added to the combined and filtered extract.

114

The extract containing internal standard was evaporated at 50 °C under a stream of

115

nitrogen. The total wax extraction was repeated in triplicate for each sample. Extracts

116

were

117

bis-N,O-(trimethylsilyl)trifluoroacetamide (BSTFA; Alfa) for 40 min at 70 °C. Then,

118

the remaining BSTFA was evaporated under a stream of nitrogen, and each sample

119

was re-dissolved in chloroform. Wax compounds were analyzed by a Finnigan gas

120

chromatograph (Trace GC Ultra) equipped with a DB-1 MS capillary column (30 m ×

121

0.25 mm, i.d. ×0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and attached

122

to a Finnigan mass spectrometer (Trace DSQ). GC-MS conditions were as follows:

123

column temperature, 2 min at 70 °C, increase from 70 °C to 200 °C at a rate of

incubated

Analysis of

in

Cuticular Wax. Wax extraction and

100

µL

pyridine

7

ACS Paragon Plus Environment

and

100

gas

µL

Journal of Agricultural and Food Chemistry

124

20 °C/min, 2 min at 200 °C, increase from 200 °C to 310 °C at a rate of 3 °C/min, and

125

30 min at 310 °C; injection at 250 °C; MS transfer line at 250 °C; ion source at

126

270 °C; carrier gas, helium (1.0 mL/min); 10:1 split ratio; EI 70 eV; and m/z 50–650.

127

Wax compounds were identified either by the NIST11 MS Library or by comparing

128

their mass spectra and retention times with those of generic standards. Triterpenoids

129

were quantified using the external standard method based on calibration curves, while

130

VLC aliphatic compounds were quantified by comparison with known amounts of the

131

internal standard tetracosane (17). The results were expressed as an absolute (per unit

132

of fruit surface area, µg/cm2) and relative (the percentage of each compound

133

compared to the total wax content, %) value.

134

Determination of Quality Parameters. Twenty blueberry fruits were randomly

135

selected to measure the surface color (lightness, L*; change of greenness to redness, a*;

136

and change of blueness to yellowness, b*) using the chromameter CR-400 (Konica

137

Minolta Sensing, Tokyo, Japan). Firmness of blueberry fruit was measured using a

138

TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, U.K.) with a

139

5 mm diameter stainless steel probe. Each fruit which peel was removed before

140

determination was equatorially compressed by 5 mm distance at a speed of 1 mm/s

141

with a 5-g trigger force, and the maximum force (N) was recorded. The weight loss 8

ACS Paragon Plus Environment

Page 8 of 32

Page 9 of 32

Journal of Agricultural and Food Chemistry

142

was determined by weighing 100 fruits before and after the storage period and

143

expressed as a percentage of the initial weight. The decay incidence was evaluated by

144

the number of decayed fruits relative to the total number of fruits (100 fruits). Fruits

145

with visible fungal growth or bacterial lesions on the surface were considered to be

146

decayed.

147

Total soluble solids (TSS) and titratable acidity (TA) were assessed in fruit juice

148

obtained from 20 fruits. TSS was determined using a digital hand-held refractometer

149

(Atago PAL-1, Tokyo, Japan). TA was determined by titration with 0.05 M NaOH to

150

an end-point pH of 8.2, and the results were expressed as a percentage of citric acid.

151

Statistical Analysis. All experiments were performed at least in triplicate, and the

152

results were expressed as means ± standard deviation. One-way analysis of variance

153

(ANOVA) in conjunction with Duncan’s multiple-range test was performed to

154

identify statistically significant differences among the treatment levels at P < 0.05. All

155

analyses were carried out using SPSS 21 (IBM, Armonk, NY, USA).

156 157

RESULTS AND DISCUSSION

158

Changes in Fruit Quality and Wax Content during Ripening. The quality

159

parameters and cuticular wax contents of ‘Legacy’ and ‘Brightwell’ blueberries at 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

160

three different phonological stages are shown in Table 1. The average weight,

161

diameter, surface area per fruit, a*, TSS and TSS/TA significantly (P < 0.05)

162

increased, whereas L*, b*, firmness and TA significantly (P < 0.05) decreased, with

163

fruit ripening in both cultivars. The amounts of cuticular wax increased by 38.61% in

164

‘Legacy’ blueberries and by 23.43% in ‘Brightwell’ blueberries from T1 to T3. The

165

total wax content increased continuously during fruit ripening. This observation was

166

supported by in the findings in several previous studies of orange (11), tomato (19)

167

and pear (20). Within each phenological stage, no significant differences were

168

observed in TSS, TA, and the tested physical parameters including average weight,

169

diameter, surface area per fruit and skin color, between the two cultivars, whereas the

170

total wax content in ‘Brightwell’ blueberries was 2-fold greater than that in ‘Legacy’

171

blueberries.

172

Changes in Wax Composition during Ripening. Triterpenoids. Triterpenoids are

173

the primary cuticular wax compounds in grape (16), peach (21) and blueberry (9,17).

174

As shown in Figure1, a significant (P < 0.05) increase in the content of triterpenoids

175

was observed from T1 to T3 in both cultivars, suggesting that the biosynthesis of

176

triterpenoids occurred during fruit ripening. However, changes in the relative content

177

of triterpenoids were different between the two cultivars during fruit ripening. The

10

ACS Paragon Plus Environment

Page 10 of 32

Page 11 of 32

Journal of Agricultural and Food Chemistry

178

relative content of triterpenoids did not change significantly from T1 to T3 in the

179

‘Legacy’ blueberries but increased significantly (P < 0.05) in the ‘Brightwell’

180

blueberries (Figure 2A). The relative content of triterpenoid acids increased

181

significantly (P < 0.05), whereas that of triterpenoid alcohols decreased significantly

182

(P < 0.05), during fruit ripening in both cultivars (Table 2). The deposition pattern of

183

triterpenoids in cuticular wax differs among fruit species due to genetic and

184

environmental factors (15). Peschel and others (22) reported that the content of

185

triterpenes decreased during fruit ripening in sweet cherry. A decrease in the content

186

of triterpenoids was also observed during fruit ripening in grape (16). However,

187

triterpenoids continuously accumulated in cuticular wax during fruit ripening in

188

tomato (19,23) and orange (24). We found that the contents of triterpenoids increased

189

significantly (P < 0.05) during fruit ripening in both blueberry cultivars (Figure 1);

190

and the primary triterpenoid was oleanolic acid in ‘Legacy’ blueberries and ursolic

191

acid in ‘Brightwell’ blueberries (Table 2).

192

VLC aliphatic compounds. VLC aliphatic compounds, including primary alcohols,

193

alkyl esters, aldehydes, alkanes, secondary alcohols, ketones and fatty acids, have

194

chain lengths ranging from C20 to C34 (14). As shown in Figure 1 and Table 2,

195

diketones, aldehydes, primary alcohols, fatty acids and alkanes were identified in the

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

196

cuticular wax of both cultivars, and the dominant VLC aliphatic compounds were

197

β-diketones. However, hentriacontane-10,12-dione was found only in ‘Legacy’

198

blueberries and tritriacontane-12,14-dione was found only in ‘Brightwell’ blueberries.

199

No significant differences were identified in the absolute content of diketones among

200

the three different phenological stages in both cultivars (Figure 1), whereas the

201

relative content of diketones decreased significantly (P < 0.05) from T1 to T3 (Figure

202

2B). The content of aldehydes, primary alcohols, fatty acids and alkanes increased

203

from T1 to T3 in both cultivars (Figure 1), indicating that these wax compounds

204

gradually accumulated during blueberry fruit ripening. However, a different

205

deposition rate was found among the different wax classes during fruit ripening. In

206

‘Legacy’ blueberries, the relative content of aldehydes, primary alcohols and fatty

207

acids increased by 2.71-fold (from 2.616 to 7.092%), 4.75-fold (from 1.036 to

208

4.917%), and 2.66-fold (from 1.319 to 3.511%), respectively, from T1 to T3, whereas

209

the relative content of alkanes decreased by 1.17-fold (from 1.726 to 1.475%). In

210

‘Brightwell’ blueberries, the relative content of all VLC compounds showed different

211

fold increases from T1 to T3 (Figure 2C–F).

212

Changes in Fruit Quality and Wax Content during Cold Storage. Changes in the

213

quality attributes of ‘Legacy’ and ‘Brightwell’ blueberries harvested at T3 during cold

12

ACS Paragon Plus Environment

Page 12 of 32

Page 13 of 32

Journal of Agricultural and Food Chemistry

214

storage are shown in Table 3. The average weight, diameter and surface area per fruit

215

of both cultivars did not change significantly after 30 d of storage at 4 °C. However,

216

the total wax content decreased significantly (P < 0.05) with storage times in both

217

cultivars, and similar results were obtained in previous studies involving sweet cherry

218

(6), Asian pear (12) and apple (25).

219

The weight loss increased significantly (P < 0.05) with the increasing storage time

220

in both cultivars; however, it was more in ‘Brightwell’ blueberries than in ‘Legacy’

221

blueberries (Table 3). To a large extent, the postharvest weight loss in fruits is

222

attributed to water loss, and postharvest water loss in fruits is restricted by cuticular

223

wax (26). However, the postharvest water loss is not related to the total wax content

224

whereas closely related to lipid composition of the cuticle (27,28). Previous studies

225

showed that the high ratio of n-alkanes to triterpenoids in cuticular wax decreases the

226

water loss rate during postharvest storage of pepper, peach and sweet cherry (6,21,27).

227

Moggia and others (9) also found that ursolic acid content in cuticular wax was highly

228

correlated with weight loss in the blueberry cultivars ‘Duke’ and ‘Brigitta’. We found

229

that weight loss was negatively correlated with the ratio of n-alkanes to triterpenoids

230

(r = -0.82) in ‘Legacy’ blueberries and also with the contents of primary alcohols and

231

fatty acids (r = -0.96) in ‘Brightwell’ blueberries. In blueberries, the intracuticular

13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

232

wax layer, which is composed of aliphatic compounds (29), functions as a

233

transpiration barrier and thus, the weight loss is determined by the content of alkanes,

234

primary alcohols and fatty acids.

235

Cuticular wax is one of the major barriers against fungal infections and can inhibit

236

the conidial germination and mycelial growth of some fungi such as Alternaria

237

alternata (20). Gabler and others (30) reported that wax concentration was positively

238

correlated with resistance to mold in grape. We found that the decay incidence

239

increased significantly (P < 0.05) after 15-30 d of storage in both cultivars; however,

240

the decay incidence was 40.43% lower in ‘Brightwell’ blueberries than that in

241

‘Legacy’ blueberries after 30 d of storage. This may be partially due to the higher

242

cuticular wax content in ‘Brightwell’ blueberries (Table 3).

243

Changes in Wax Composition during Postharvest Cold Storage. Changes in the

244

cuticular wax composition of ‘Legacy’ and ‘Brightwell’ blueberries during cold

245

storage are shown in Figure3 and Table 4. Two triterpenoid alcohols, amyrin and

246

lupeol, and two triterpenoid acids, oleanolic and ursolic acids, were identified in the

247

relative content of triterpenoids in both cultivars. However, large differences were

248

detected between ‘Legacy’ and ‘Brightwell’ blueberries for all of the identified

249

compounds. Similar results were also reported by Moggia and others (9). During cold

14

ACS Paragon Plus Environment

Page 14 of 32

Page 15 of 32

Journal of Agricultural and Food Chemistry

250

storage, the absolute and relative content of triterpenoids did not change significantly

251

in ‘Legacy’ blueberries. However, the absolute content of triterpenoids decreased

252

from 123.01 µg/cm2 to 108.36 µg/cm2, whereas the relative content increased from

253

68.69 to 73.67% after 30 d of storage in ‘Brightwell’ blueberries. This was probably

254

due to a significant (P < 0.05) increase in the relative amount of ursolic acid.

255

Cultivar-related differences have also been observed in the content of triterpenoids in

256

cuticular wax during postharvest storage in peach and sweet cherry (6,21).

257

A total of 34 aliphatic compounds, including diketones (15.44–18.77%), aldehydes

258

(C24-C32, 1.36–7.09%), primary alcohols (C22-C30, 2.25–4.92%), fatty acids

259

(C16-C30, 1.69–3.51%), and alkanes (C23-C31, 0.89–1.47%), were detected in both

260

cultivars (Supporting information; Table 4). In ‘Legacy’ blueberries, the content of

261

diketones decreased from 16.43 µg/cm2 to 14.10 µg/cm2 during the entire 30 d storage

262

period. No significant change was identified in the content of other aliphatic

263

compounds during cold storage (Figure 3). In ‘Brightwell’ blueberries, significant

264

decreases in the contents of aliphatic compounds were observed during the tested 30 d

265

storage period. Specifically, the contents of diketones, aldehydes, primary alcohols,

266

fatty acids and alkanes decreased by 34.6, 36.36, 32.4, 37.9 and 22.4%, respectively

267

(Figure 3).

15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

268

Overall, our results showed that cuticular wax was gradually deposited on the

269

epidermis of blueberry during fruit ripening, and played a vital role in postharvest

270

storage quality of blueberries possibly due to reducing the water loss and infection

271

susceptibility. The major wax compounds included triterpenoids, aldehydes, fatty

272

acids, primary alcohols and alkanes. Also noted was that the total wax contents and

273

compounds might differ in different blueberry cultivars and decrease during storage.

274

The results not only provided aquantitative and qualitative overview of cuticular wax

275

compounds of blueberries at different phenological stages and postharvest storage

276

periods, but also warranted additional research in investigate how wax components

277

may be accumulated during blueberry ripening, and how wax components may

278

contribute to postharvest storage quality of blueberries.

279 280

ACKNOWLEDGEMENT

281

This work was supported by the National Natural Science Foundation of China (Grant

282

No. 31772042, 31471635), the National Science & Technology Support Program of

283

China (Grant No. 2015BAD16B06) and the National Special Fund for Agro-scientific

284

Research in the Public Interest, China (Grant No.201303073).

285 16

ACS Paragon Plus Environment

Page 16 of 32

Page 17 of 32

Journal of Agricultural and Food Chemistry

286

Supporting Information.

287

Table S1 and S2 list the wax constituents (relative %) identified on ‘Legacy’ and

288

‘Brightwell’ blueberries at phenological stage and after cold storage, respectively.

289

This material is available free of charge via the Internet at http://pubs.acs.org.

290 291 292 293

REFERENCES (1) Koca, I.; Karadeniz, B. Antioxidant properties of blackberry and blueberry fruits grown in the Black Sea Region of Turkey. Sci. Hortic. 2009, 121, 447-450.

294

(2) Reque, P. M.; Steckert, E. C.; dos Santos, F. T.; Danelli, D.; Jablonski, A.;

295

Flôres, S. H.; Rech, R.; de O. Rios, A.; de Jong, E. V. Heat processing of blueberries

296

and its effect on their physicochemical and bioactive properties. J. Food Process Eng.

297

2016, 39, 564-572.

298

(3) Shi, M.; Loftus, H.; McAinch, A. J.; Su, X. Q. Blueberry as a source of

299

bioactive compounds for the treatment of obesity, type 2 diabetes and chronic

300

inflammation. J. Funct. Foods 2017, 30, 16-29.

301 302

(4) Samuels, L.; Kunst, L.; Jetter, R. Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu. Rev. Plant Biol. 2008, 59, 683-707.

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

303 304

(5) Shepherd, T.; Griffiths, D. W. The effects of stress on plant cuticular waxes. New Phytol. 2006, 171, 469-499.

305

(6) Belge, B.; Llovera, M.; Comabella, E.; Gatius, F; Guillen, P.; Graell, J.; Lara, I.

306

Characterization of cuticle composition after cold storage of "Celeste" and "Somerset"

307

sweet cherry fruit. J. Agric. Food. Chem. 2014, 62, 8722-8729.

308

(7) Rios, J. C.; Robledo, F.; Schreiber, L.; Zeisler, V.; Lang, E.; Carrasco, B.;

309

Silva, H. Association between the concentration of n-alkanes and tolerance to

310

cracking in commercial varieties of sweet cherry fruits. Sci. Hortic. 2015, 197, 57-65.

311

(8) Mukhtar, A.; Damerow, L.; Blanke, M. Non-invasive assessment of glossiness

312

and polishing of the wax bloom of European plum. Postharvest Biol. Technol. 2014,

313

87, 144-151.

314

(9) Moggia, C.; Graell, J.; Lara, I.; Schmeda-Hirschmann, G.; Thomas-Valdés, S.;

315

Lobos, G. A. Fruit characteristics and cuticle triterpenes as related to postharvest

316

quality of highbush blueberries. Sci. Hortic. 2016, 211, 449-457.

317

(10) Wisuthiphaet, N.; Damerow, L.; Blanke, M. M. Non-destructive detection of

318

the wax bloom on European plum during post-harvest handling. J. Food Eng. 2014,

319

140, 46-51.

18

ACS Paragon Plus Environment

Page 18 of 32

Page 19 of 32

Journal of Agricultural and Food Chemistry

320

(11) Liu, D. C.; Zeng, Q.; Ji, Q. X.; Liu, C. F.; Liu, S. B.; Liu, Y. A comparison of

321

the ultrastructure and composition of fruits’ cuticular wax from the wild-type

322

‘Newhall’ navel orange (Citrus sinensis [L.] Osbeck cv. Newhall) and its glossy

323

mutant. Plant Cell Rep. 2012, 31, 2239-2246.

324

(12) Wu, X.; Yin, H.; Chen, Y.; Li, L.; Wang, Y.; Hao, P.; Cao, P.; Qi, K.; Zhang,

325

S. Chemical composition, crystal morphology and key gene expression of cuticular

326

waxes of Asian pears at harvest and after storage. Postharvest Biol. Technol. 2017,

327

132, 71-80.

328 329

(13) Borisjuk, N.; Hrmova, M.; Lopato, S. Transcriptional regulation of cuticle biosynthesis. Biotechnol. Adv. 2014, 32, 526-540.

330

(14) Racovita, R. C.; Hen-Avivi, S.; Fernandez-Moreno, J. P.; Granell, A.; Aharoni,

331

A.; Jetter, R. Composition of cuticular waxes coating flag leaf blades and peduncles

332

of Triticum aestivum cv. Bethlehem. Phytochem. 2016, 130, 182-192.

333

(15) Vichi, S.; Cortés-Francisco, N.; Caixach, J.; Barrios, G.; Mateu, J.; Ninot, A.;

334

Romero, A. Epicuticular wax in developing olives (Olea europaea) is highly

335

dependent upon cultivar and fruit ripeness. J. Agric. Food. Chem. 2016, 64,

336

5985-5994.

19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

337

(16) Pensec, F.; Pączkowski, C.; Grabarczyk, M.; Wozńiak, A.; Beńard-Gellon, M.;

338

Bertsch, C.; Chong, J.; Szakiel, A. Changes in the triterpenoid content of cuticular

339

waxes during fruit ripening of eight grape (Vitis vinifera) cultivars grown in the Upper

340

Rhine Valley. J. Agric. Food. Chem. 2014, 62, 7998-8007.

341

(17) Chu, W.; Gao, H.; Cao, S.; Fang, X.; Chen, H.; Xiao, S. Composition and

342

morphology of cuticular wax in blueberry (Vaccinium spp.) fruits. Food Chem. 2017,

343

219, 436-442.

344 345

(18) Chu, W.; Gao, H.; Chen H.; Fang, X.; Zheng Y. Effects of cuticular wax on the postharvest quality of blueberry fruit. Food Chem. 2018, 239, 68-74.

346

(19) Leide, J.; Hildebrandt, U.; Reussing, K.; Riederer, M.; Vogg, G. The

347

developmental pattern of tomato fruit wax accumulation and its impact on cuticular

348

transpiration barrier properties: effects of a deficiency in β-ketoacyl-coenzyme A

349

synthase (LeCER6). Plant Physiol. 2007, 144, 1667-1679.

350

(20) Li, Y.; Yin, Y.; Chen, S.; Bi, Y.; Ge, Y. Chemical composition of cuticular

351

waxes during fruit development of Pingguoli pear and their potential role on early

352

events of Alternaria alternata infection. Funct. Plant Biol. 2014, 41, 313-320.

20

ACS Paragon Plus Environment

Page 20 of 32

Page 21 of 32

Journal of Agricultural and Food Chemistry

353

(21) Belge, B.; Llovera, M.; Comabella, E.; Graell, J.; Lara, I. Fruit cuticle

354

composition of a melting and a nonmelting peach cultivar. J. Agric. Food. Chem.

355

2014, 62, 3488-3495.

356 357

(22) Peschel, S.; Franke, R.; Schreiber, L.; Knoche, M. Composition of the cuticle of developing sweet cherry fruit. Phytochem. 2007, 68, 1017-1025.

358

(23) Kosma, D. K.; Parsons, E. P.; Isaacson, T.; Lü, S.; Rose, J. K. C.; Jenks, M. A.

359

Fruit cuticle lipid composition during development in tomato ripening mutants.

360

Physiol. Plant. 2010, 139, 107-117.

361

(24) Wang, J.; Sun, L.; Xie, L.; He, Y.; Luo, T.; Sheng, L.; Luo, Y.; Zeng, Y.; Xu,

362

J.; Deng, X.; Cheng, Y. Regulation of cuticle formation during fruit development and

363

ripening in 'Newhall' navel orange (Citrus sinensis Osbeck) revealed by

364

transcriptomic and metabolomic profiling. Plant Sci. 2016, 243, 131-144.

365

(25) Dong, X.; Rao, J.; Huber, D. J.; Chang, X.; Xin, F. Wax composition of 'Red

366

Fuji' apple fruit during development and during storage after 1-methylcyclopropene

367

treatment. Hortic. Environ. Biotechnol. 2012, 53, 288-297.

368 369

(26) Lara, I.; Belge, B.; Goulao, L. F. The fruit cuticle as a modulator of postharvest quality. Postharvest Biol. Technol. 2014, 87, 103-112.

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

370

(27) Parsons, E. P.; Popopvsky, S.; Lohrey, G. T.; Lü, S.; Alkalai-Tuvia, S.;

371

Perzelan, Y.; Paran, I.; Fallik, E.; Jenks, M. A. Fruit cuticle lipid composition and

372

fruit post-harvest water loss in an advanced backcross generation of pepper

373

(Capsicum sp.). Physiol. Plant. 2012, 146, 15-25.

374

(28) Parsons, E. P.; Popopvsky, S.; Lohrey, G. T.; Alkalai-Tuvia, S.; Perzelan, Y.;

375

Bosland, P.; Bebeli, P. J.; Paran, I.; Fallik, E.; Jenks, M. A. Fruit cuticle lipid

376

composition and water loss in a diverse collection of pepper (Capsicum). Physiol.

377

Plant. 2013, 149, 160-174.

378

(29) Vogg, G.; Fischer, S.; Leide, J.; Emmanuel, E.; Jetter, R.; Levy, A. A.;

379

Riederer, M. Tomato fruit cuticular waxes and their effects on transpiration barrier

380

properties: functional characterization of a mutant deficient in a very-long-chain fatty

381

acid β-ketoacyl-CoA synthase. J. Exp. Bot. 2004, 55, 1401-1410.

382

(30) Gabler, F. M.; Smilanick, J. L.; Mansour, M.; Ramming, D. W.; Mackey, B. E.

383

Correlations of morphological, anatomical, and chemical features of grape berries

384

with resistance to Botrytis cinerea. Phytopathol. 2003, 93, 1263-1273.

385

386

22

ACS Paragon Plus Environment

Page 22 of 32

Page 23 of 32

Journal of Agricultural and Food Chemistry

387

Figure Captions

388

Figure 1. Content of major wax compounds identified in ‘Legacy’ (A) and

389

‘Brightwell’ (B) blueberries at three phenological stages (T1, T2, and T3). Different

390

letters indicate significant differences among phenological stages within each class of

391

wax compounds at P < 0.05.

392 393

Figure 2. Relative content of triterpenoids (A), diketones (B), aldehydes (C), primary

394

alcohols (D), fatty acids (E) and alkanes (F) in ‘Legacy’ and ‘Brightwell’ blueberries

395

at three phenological stages (T1, T2, and T3). Different letters indicate significant

396

differences among phenological stages within each cultivar at P < 0.05.

397 398

Figure 3. Content of major wax compounds identified in ‘Legacy’ (A) and

399

‘Brightwell’ (B) blueberries after 0 d, 15 d, and 30 d of storage at 4°C. Different

400

letters indicate significant differences among storage periods within each class of wax

401

compounds at P < 0.05.

402 403 404

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 24 of 32

405

Tables

406

Table 1. Quality Attributes of Fresh ‘Legacy’ and ‘Brightwell’ Blueberries at Three Phenological Stages (T1, T2 and T3) Legacy Parameter

T1

Weight per fruit (g) Diameter (mm) Fruit surface (cm2/fruit)

T3

T1

T2

T3

0.97 ± 0.13c

1.45 ± 0.10 b

1.76 ± 0.12 a

1.05 ± 0.16 c

1.52 ± 0.10 b

1.81 ± 0.11 a

11.21 ± 0.56 c

12.06 ± 0.54 b

13.08 ± 0.39 a

11.29 ± 0.61 c

12.34 ± 0.69 b

13.17 ± 0.57 a

3.96 ± 0.39 c

4.58 ± 0.41 b

5.37 ± 0.32 a

4.01 ± 0.44 c

4.79 ± 0.53 b

5.46 ± 0.47 a

43.84 ± 4.80 b 32.23 ± 1.93 d

65.03 ± 3.96 a

46.29 ± 4.84 b

35.01 ± 2.95 c

*

L

62.96 ± 4.22 a

a*

-11.67 ± 5.10 d

11.64 ± 2.99 b

0.43 ± 0.78 c

-10.42 ± 2.80 d

14.53 ± 3.26 a

0.62 ± 1.77 c

*

28.85 ± 4.73 a

2.05 ± 3.54 c

-5.03 ± 0.84 d

24.50 ± 3.12 b

2.96 ± 3.12 c

-4.63 ± 0.89 d

Firmness (N)

5.12 ± 0.61 b

2.57 ± 0.36 c

1.20 ± 0.21 d

5.58 ± 1.00 a

2.89 ± 0.35 c

1.36 ± 0.21 d

o

TSS ( Brix)

8.17 ± 0.25 c

9.33 ± 0.25 b

11.63 ± 0.15 a

8.13 ± 0.31 c

9.70 ± 0.20 b

11.97 ± 0.21 a

TA (%)

2.42 ± 0.04 a

1.89 ± 0.07 b

0.59 ± 0.01 c

2.44 ± 0.09 a

1.84 ± 0.05 b

0.57 ± 0.01 c

3.37 ± 0.08 d

4.95 ± 0.13 c 19.61 ± 0.22 b

3.34 ± 0.14 d

5.27 ± 0.21 c

21.10 ± 0.72 a

145.12 ± 10.39 b 164.63 ± 8.45 a

179.10 ± 4.83 a

b

TSS/TA 2

Total wax content (µg/cm )

407

T2

Brightwell

63.17 ± 3.44 d 73.65 ± 1.96 cd

87.60 ± 4.60 c

TSS, total soluble solids; TA, titratable acidity. Different letters indicate significant differences within each row at P < 0.05.

408

24

ACS Paragon Plus Environment

Page 25 of 32

Journal of Agricultural and Food Chemistry

409

Table 2. Relative Content (%) of Triterpenoids and Diketones in Fresh ‘Legacy’ and ‘Brightwell’ Blueberries at Three Phenological Stages (T1,

410

T2 and T3)a Legacy

Brightwell

T1

T2

T3

T1

T2

T3

β-amyrin

16.772 ± 1.176 a

12.526 ± 1.241 b

11.125 ± 0.294 b

4.474 ± 0.122 a

3.811 ± 0.250ab

3.666 ± 0.232 b

α-amyrin

2.145 ± 0.258 a

1.846 ± 0.158 a

1.954 ± 0.114 a

10.399 ± 0.058 a

9.082 ± 0.267 b

8.763 ± 0.250 b

Lupeol

5.782 ± 0.159 a

5.482 ± 0.586 a

4.031 ± 0.185 b

4.601 ± 0.186 a

3.783 ± 0.287ab

2.969 ± 0.346 b

26.932 ± 0.591 b

28.740 ± 0.401 a

29.845 ± 0.013 a

9.530 ± 0.243 b

10.736 ± 0.369 a

10.922 ± 0.313 a

5.551 ± 0.330 b

8.470 ± 0.994 a

8.881 ± 0.029 a

37.999 ± 0.812 c

40.368 ± 0.604 b

42.372 ± 0.216 a

Hentriacontane-10,12-dione

24.896 ± 0.522 a

20.423 ± 1.611 b

18.775 ± 0.570 b

nd

Tritriacontane-12,14-dione

nd

nd

nd

19.468 ± 0.495 a

Triterpenoids

Oleanolic acid Ursolic acid

Diketones nd 17.138 ± 0.597 b

nd 15.444 ± 0.458 c

411

a

412

means ± standard deviation of three replicates. Different letters indicate significant differences within each row and cultivar at P < 0.05.

Relative content (%) was regarded as percentage of each compound compared to the total wax content. nd, non-detectable. Values represent

413

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 32

414 415

Table 3. Quality Attributes of ‘Legacy’ and ‘Brightwell’ Blueberries after 0 d, 15 d and 30 d of Storage at 4 °C Parameter

Legacy

Brightwell

0d

15 d

Weight per fruit (g)

1.76 ± 0.12 ab

1.74 ± 0.10 ab

1.71 ± 0.10 b

1.81 ± 0.11 a

1.79 ± 0.11 a

1.75 ± 0.11 ab

Diameter (mm)

13.08 ± 0.39 a

13.11 ± 0.41 a

12.95 ± 0.54 a

13.17 ± 0.57 a

13.12 ± 0.55 a

12.89 ± 0.44 a

5.37 ± 0.32 a

5.40 ± 0.34 a

5.27 ± 0.44 a

5.46 ± 0.47 a

5.41 ± 0.46 a

5.22 ± 0.36 a

87.60 ± 4.60 c

65.61 ± 6.17 d

80.41 ± 5.71 cd

179.10 ± 4.83 a

138.23 ± 9.32 b

147.07 ± 6.10 b

Weight loss (%)

-

1.55 ± 0.12 d

3.80 ± 0.12 b

-

1.86 ± 0.09 c

4.19 ± 0.18 a

Decay incidence (%)

-

3.82 ± 1.29 c

15.7 ± 1.58 a

-

2.85 ± 1.26 c

11.18 ± 1.83 b

Fruit surface (cm2/fruit) 2

Total wax content (µg/cm )

30 d

0d

15 d

30 d

416

Different letters indicate significant differences within each row at P < 0.05. Blueberries used in this storage study were harvested at T3

417

phenological stage.

418

419

26

ACS Paragon Plus Environment

Page 27 of 32

420

Journal of Agricultural and Food Chemistry

Table 4. Relative Content (%) of Wax Compounds in ‘Legacy’ and ‘Brightwell’ Blueberries after 0 d, 15 d and 30 d of Storage at 4 °Ca Legacy

Brightwell

0d

15 d

30 d

Triterpenoids

55.837 ± 0.323 a

55.421 ± 0.411 a

56.987 ± 0.955 a

β-amyrin

11.125 ± 0.294 a

10.965 ± 0.492 a

11.766 ± 0.826 a

3.666 ± 0.232 a

3.548 ± 0.141 a

4.013 ± 0.057 a

α-amyrin

1.954 ± 0.114 a

1.291 ± 0.074 b

1.699 ± 0.073 a

8.763 ± 0.250 a

8.597 ± 0.104 a

9.273 ± 0.313 a

Lupeol

4.031 ± 0.185 a

3.099 ± 0.476 a

3.678 ± 0.879 a

2.969 ± 0.346 a

2.698 ± 0.120 a

3.133 ± 0.077 a

29.845 ± 0.013 a

31.404 ± 0.733 a

30.846 ± 1.245 a

10.922 ± 0.313 a

10.842 ± 0.615 a

10.491 ± 0.508 a

Ursolic acid

8.881 ± 0.029 a

8.662 ± 0.101 a

8.998 ± 0.415 a

42.372 ± 0.216 b

46.162 ± 0.935 a

46.756 ± 0.265 a

Diketones

18.775 ± 0.570 a

18.200 ± 0.744 a

17.581 ± 1.206 a

15.444 ± 0.458 a

13.020 ± 0.252 b

12.300 ± 0.371 b

Hentriacontane-10,12-dione

18.775 ± 0.570 a

18.200 ± 0.744 a

17.581 ± 1.206 a

nd

nd

nd

Tritriacontane-12,14-dione

nd

nd

nd

15.444 ± 0.458 a

13.020 ± 0.252 b

12.300 ± 0.371 b

Oleanolic acid

0d

15 d

68.692 ± 0.665 b 71.847 ± 1.467 ab

30 d 73.667 ± 0.691 a

Aldehydes

7.092 ± 0.446 a

6.160 ± 0.243 a

6.178 ± 0.241 a

1.359 ± 0.088 a

1.073 ± 0.097 b

1.056 ± 0.049 b

Primary alcohols

4.917 ± 0.392 a

5.210 ± 0.061 a

4.957 ± 0.442 a

2.247 ± 0.219 a

2.419 ± 0.244 a

1.856 ± 0.185 a

Fatty acids

3.511 ± 0.166 a

3.538 ± 0.251 a

3.227 ± 0.094 a

1.695 ± 0.240 a

1.481 ± 0.095 a

1.286 ± 0.046 a

Alkanes

1.475 ± 0.033 a

1.483 ± 0.049 a

1.417 ± 0.062 a

0.893 ± 0.039 a

0.812 ± 0.020 a

0.844 ± 0.011 a

421

a

422

means ± standard deviation of three replicates. Different letters indicate significant differences within each cultivar at P < 0.05. Blueberries used

Relative content (%) was regarded as percentage of each compound compared to the total wax content. nd, non-detectable. Values represent

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

423

in this storage study were harvested at T3 phenological stage.

28

ACS Paragon Plus Environment

Page 28 of 32

Page 29 of 32

Journal of Agricultural and Food Chemistry

424

Figures

425 426 427 428 429 430 431

Figure 1.

432 433 434 435 436 437 438 439 440 441

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

442 443 444 445 446 447 448 449 450 451 452 453 454

Figure 2.

455 456 457 458 459

30

ACS Paragon Plus Environment

Page 30 of 32

Page 31 of 32

Journal of Agricultural and Food Chemistry

460 461 462 463 464 465 466

Figure 3.

467 468 469

31

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

55x44mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 32 of 32