Accepted Manuscript

Title: The effects of fruit bagging on levels of phenolic

compounds and expression by anthocyanin biosynthetic and
regulatory genes in red-fleshed apples

Author: Xiaoqian Wang Zhiwei Wei Fengwang Ma

PII: S1359-5113(15)30034-9
DOI: http://dx.doi.org/doi:10.1016/j.procbio.2015.06.024
Reference: PRBI 10468

To appear in: Process Biochemistry

Received date: 11-3-2015

Revised date: 15-6-2015
Accepted date: 29-6-2015

Please cite this article as: Wang X, Wei Z, Ma F, The effects of fruit
bagging on levels of phenolic compounds and expression by anthocyanin
biosynthetic and regulatory genes in red-fleshed apples, Process Biochemistry (2015),
http://dx.doi.org/10.1016/j.procbio.2015.06.024

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 1 The effects of fruit bagging on levels of phenolic compounds and expression by

2 anthocyanin biosynthetic and regulatory genes in red-fleshed apples

3 Xiaoqian Wang, Zhiwei Wei, Fengwang Ma*

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5 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture,

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6 Northwest A&F University, Yangling, Shaanxi 712100, China

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7

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8 *corresponding author

9 Fengwang Ma

10 Tel: +86-29-87082648
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11 Fax: +86-29-87082648

12 E-mail: fwm64@sina.com & fwm64@nwsuaf.edu.cn

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13
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13 Abstract

14 Apples with red fruit flesh are desirable for breeding because they contain more

15 health-beneficial compounds. That colour trait is also considered more attractive to

16 consumer. To obtain experimental evidence about the molecular mechanism for this

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17 pigmentation, we investigated how the practice of bagging fruit might affect levels of

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18 phenolic compounds as well as the expression of anthocyanin biosynthetic and

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19 regulatory genes. Two red-fleshed varieties from Xinjiang, P.R. China, were studied --

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20 ‘Xiahongrou’ and ‘No.1 Hongxun’. HPLC analysis showed that the contents of

21 anthocyanins in both peel and flesh were decreased by bagging, but were increased

22
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again when fruits were re-exposed to sunlight. Except for F3H in ‘No.1 Hongxun’,
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23 transcription levels for most anthocyanin biosynthetic genes in the flesh were

24 enhanced after bag removal. MYB10 is a key transcriptional factor in the anthocyanin
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25 pathway of these two apple varieties, particularly in the flesh. These results suggest
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26 that the development of red colouring when bags are removed could be a result of
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27 light-induced coordinated expression by MYB10 and anthocyanin biosynthetic genes

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28 in red-fleshed apples.
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29 Keywords: Red-fleshed apples, Phenolics, Anthocyanins, Bagging treatment, Gene

30 expression

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31 1. Introduction

32 Phenolics in fruits and vegetables have gained much attention because of their

33 antioxidant activities and their beneficial implications for human health. This is based

34 on research demonstrating their biological activity in preventing cancer and

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35 cardiovascular diseases [1]. Apples are one of the most important dietary sources of

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36 phenolic compounds because their consumption is widespread and they are available

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37 year-round in markets. Five major polyphenolic groups are found in various apple

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38 varieties: hydroxycinnamic acids, dihydrochalcones, flavanols, flavonols, and

39 anthocyanins [2].

40
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Apple colour is a key factor when considering consumer appeal, with red fruits
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41 being the most popular [3]. This skin colour is derived from anthocyanins that belong

42 to a class of flavonoids. Though not as common, red flesh colour is a highly desirable
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43 trait that can be found with varieties such as ‘Aerlies Red Flesh’ (syn. ‘Hidden Rose’,
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44 USA), ‘Baya Marisa’ (Germany), ‘Red Devil’ (United Kingdom), and ‘Redlove’
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45 (Switzerland). This trait is explained by their genealogy [4]. The ancestor of many
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46 red-fleshed genotypes is the wild species Malus niedzwetzkyana [5], a native of

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47 Kazakhstan, Kyrgyzstan, Tajikistan, Uzbekistan, Turkmenistan, and China's Xinjiang

48 Province [6]. These varieties have high anthocyanin contents that create a dramatic

49 phenotype with strongly pigmented vegetative, floral, and fruit tissues [7].

50 Anthocyanins are synthesized from phenylalanine via the phenylpropanoid and

51 flavonoid pathway (Figure 1) [8]. The enzymes involved in this pathway include

52 phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavanone

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53 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase

54 (ANS), and UDP-glucose flavonoid 3-O-glucosyl transferase (UFGT) [9]. Its three

55 branches include flavonol synthase (FLS), which produces flavonols, as well as

56 leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), which

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57 respectively synthesize trans- and cis- flavan-3-ols, the precursors of

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58 proanthocyanidin polymers [8]. Anthocyanin biosynthesis is positively correlated with

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59 the degree of gene expression, and is controlled at the transcriptional level [10].

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60 Moreover, genes for anthocyanin production are regulated by the MYB-bHLH-WD40

61 protein complex [11]. The MYB genes (MYB1, MYB10, and MYBA) and the bHLH

62
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genes (bHLH3 and bHLH33) have been isolated in apple and shown to induce the
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63 accumulation of anthocyanins [12]. MYB1/A are mainly involved in controlling the

64 red colouration of the peel while MYB10 is responsible for colouring of the whole
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65 fruit as well as foliage [13]. In addition to genetic components, external factors, e.g.,
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66 light, temperature, mineral nutrition, and orchard management practices, can affect
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67 apple anthocyanin biosynthesis, with light being the most essential player [3,14].
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68 Red-fleshed apples are receiving increased attention because of their remarkable

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69 anthocyanin contents. Previous work with those types has largely focused on isolation

70 and functional analysis of the MYB transcription factors (TFs) [7,10,15,16]. However,

71 no reports to date have evaluated anthocyanin biosynthesis and transcriptional levels

72 in red-fleshed apples in response to light exposure, especially in the flesh. Recent

73 studies have shown that bagging treatment is an effective practice when investigating

74 the influence of light on anthocyanin synthesis in fruit [17,18]. It is also a useful

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75 technique for examining anthocyanin biosynthesis and gene expression in apples [3].

76 Therefore, we used two red-fleshed Xinjiang varieties -- ‘Xiahongrou’ and ‘No.1

77 Hongxun’ -- to monitor how bagging might change the levels of anthocyanins and

78 phenolic compounds as well as the expression of key anthocyanin biosynthetic and

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79 regulatory genes. We proposed that our findings would be helpful in improving our

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80 understanding of the molecular mechanism for red pigmentation in apple flesh.

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81

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82 2. Materials and methods

83 2.1. Plant materials and experimental treatments

84
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Red-fleshed apples ‘Xiahongrou’ and ‘No.1 Hongxun’ (two Xinjiang varieties)
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85 were studied (Figure 2). Both are within Malus niedzwetzkyana. The 6~8-year-old

86 trees were grafted and grown on M. sieversii rootstocks, and were approximately 3.5
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87 m tall with a central leader at a spacing of 2×4 m. They were cultured in an orchard at
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88 the Horticultural Experimental Station of Northwest A&F University, Yangling (34°20

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89 N, 108°24 E), China, where they received standard horticultural treatment, including
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90 pest and disease control.

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91 Two trees were used for each variety. 80 well-exposed fruits per tree were selected

92 for the control group, and the rest (80~100 fruits) were bagged with light

93 impermeable double layer (the outer layer is yellow outside and black inside, and the

94 inner layer is red) paper bags. The bags were placed on designated young fruit

95 approximately six weeks after bloom. Harvest time was determined by the starch

96 index. As the past experience, all bags were removed 15 d prior to harvest at afternoon

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 97 and the fruit was completely re-exposed to light for the remainder of the experimental

98 period. The peel and flesh were separated and collected at 0, 3, 6, 9, and 15 d after the

99 bag removal. Control samples were also collected on those days. Each treatment was

100 repeated three times in a completely randomized design. Each point-in-time sample

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101 consisted of a total of ten fruits per replicate (five fruits per tree). All samples were

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102 immediately frozen in liquid nitrogen and stored at –80°C.

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103 2.2. Analysis of phenolic compounds

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104 Flesh and peel tissues were first ground separately to fine powders in liquid

105 nitrogen and mixed well to ensure representative sampling of each variety for each

106
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sampling date. Their phenolic compounds were then extracted and analysed as
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107 described by Zhang et al. (2010) [19]. Briefly, the compounds were extracted at 0°C

108 with 70% methanol containing 2% formic acid and analysed using an Agilent 1200
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109 Liquid Chromatograph equipped with a diode array detector (Agilent Technology,
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110 Palo Alto, CA, USA). An Inertsil ODS-3 column (5.0 µm particle size, 4.6 mm×250
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111 mm; GL Sciences Inc., Tokyo, Japan) was used in the separation, preceded by an
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112 Inertsil ODS-3 Guard Column (5.0 µm, 4.0 mm×10.0 mm; GL Sciences Inc., Tokyo,
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113 Japan). Solvent A consisted of 10% formic acid (11.36% of 88% formic acid) in water

114 while solvent B was 10% formic acid and 1.36% water (11.36% of 88% formic acid)

115 in acetonitrile. The gradient followed 95% A (0 min), 85% A (25 min), 78% A (42

116 min), 64% A (60 min), and 95% A (65 min). Post-run time was 10 min. Simultaneous

117 monitoring was performed at 280 nm for catechin, epicatechin, procyanidin B1,

118 procyanidin B2, phloridzin, gallic acid, and syringic acid; at 320 nm for chlorogenic

Page 6 of 35
119 acid, caffeic acid, ρ-coumaric acid, and ferulic acid; at 365 nm for

120 quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-rhamnoside,

121 quercetin-3-rutinoside, quercetin-3-xyloside, and quercetin-3-arabinoside; and at 520

122 nm for cyanidin-3-galactoside and cyanidin-3-glucoside. Peaks were identified by

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123 comparing retention times and UV spectra with authentic standards. Contents of

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124 individual phenolic compounds were determined based on peak areas and calibration

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125 curves derived from corresponding authentic phenolic compounds.

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126 2.3. Expression analysis by quantitative real-time PCR

127 We used qRT-PCR to measure the transcript levels of key genes involved in

128
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anthocyanin biosynthesis. Total RNA was extracted from the peel and flesh samples
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129 by the Cetyltrimethyl Ammonium Bromide (CTAB) method [20]. First-strand cDNA

130 was synthesized with a PrimeScript RT Reagent Kit (Takara Bio Inc., Tokyo, Japan)
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131 according to the manufacturer's protocol. After dilution to 150 ng/μL, quantitative
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132 real-time expression was evaluated (three technical replicates) on an iQ5.0 instrument
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133 (Bio-Rad Laboratories, Hercules, CA, USA) using a SYBR Premix Ex Taq II (Tli
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134 RNaseH Plus) kit (Takara Bio Inc., Tokyo, Japan). The program included 95°C for 3
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135 min, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. A

136 melting curve was generated for each sample at the end of each run to ensure the

137 purity of the amplified products. Primers were those described by Wang et al. (2013)

138 [12] for Actin (AB638619), CHS (AB074485), F3H (AB074486), DFR (AF117268),

139 ANS (AB074487), and UFGT (AF117267); by Telias et al. (2011) [21] for MYB10

140 (DQ267896), bHLH3 (CN934367), and bHLH33 (DQ266451); by Li et al. (2007) [22]

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141 for FLS (AF119095), LAR (AY830131), and ANR (AY830130); and by Feng et al.

142 (2014) [18] for PAL (MDP0000139075). Actin served as the internal control. Data

143 were analysed by iQ5 2.0 software (Bio-Rad), using the ddCT method.

144 2.4. Statistical analysis

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145 All data were analysed with SPSS 17.0. Independent-samples t-test were used to

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146 determine the significance of differences among samples at a level of 0.05. Values

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147 were reported as means ± standard deviation (SD) from triplicate experiments.

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148

149 3. Results and discussion

150
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3.1. Effects of bagging on the contents of phenolic compounds
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151 Changes in levels of phenolics within red-fleshed apple extracts in response to

152 bagging are presented in Table 1 and 2. Two types of benzoic acids and four types of
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153 hydroxycinnamic acids were quantified (data not shown for all), with chlorogenic acid
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154 being the major one. Both in the peel and flesh, no differences in chlorogenic acid
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155 contents were found between bagged and control fruit from either variety. However,
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156 the content was enhanced in the flesh of ‘Xiahongrou’. Similar results have been
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157 reported by Awad et al. (2001) [23], who showed that the amounts of chlorogenic acid

158 in apple are largely independent of light levels. After the bags were removed, changes

159 for chlorogenic acid contents differed significantly among two varieties. After 15 d of

160 re-exposure to sunlight, the chlorogenic acid contents in both peel and flesh from

161 ‘No.1 Hongxun’ exceeded those of the control, whereas those measured in

162 ‘Xiahongrou’ fruit were similar to levels found in the controls. Feng et al. (2014) [18]

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163 have reported that, after bags are removed from ‘Jonagold’ apples, those contents are

164 increased in both the peel and flesh. Therefore, it appears that changes in chlorogenic

165 acid levels are highly dependent upon the varieties that were tested.

166 Phloridzin is the dominant dihydrochalcone in apples. We found that, for both peel

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167 and flesh, bagging had very little influence on contents of that compound. The

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168 exception was a decline in the peel from ‘Xiahongrou’. Removal of the bags increased

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169 the levels of phloridzin in the fruit of the two red-fleshed varieties before Day 9,

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170 except that in the flesh of ‘Xiahongrou’. This was in accord with results reported by

171 Feng et al. (2014) [18].

172
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Within the group of flavanols, bagging significantly decreased the peel contents of
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173 procyanidin B1, catechin, and procyanidin B2 in ‘Xiahongrou’ and catechin in ‘No.1

174 Hongxun’. In the flesh, most of those flavanols were also diminished by bagging in
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175 ‘No.1 Hongxun’. However, the contents of procyanidin B1 and catechin were
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176 enhanced in ‘Xiahongrou’. Chen et al. (2012) [24] have conducted bagging
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177 experiments with three apple varieties and found that, although flavanol levels were
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178 reduced in the peel, the response in flesh samples was highly dependent upon the
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179 particular variety. We noted that, after the bags were removed, the contents of

180 flavanols overall were significantly improved in the peel and flesh of both varieties.

181 However, the opposite was found for content of catechin in the flesh of ‘Xiahongrou’.

182 After 15 d of re-exposure to sunlight, the levels of most tested flavanols in the peel

183 and flesh of previously bagged fruits reached values that were similar to or even

184 higher than those measured in the control fruits that had never been bagged.

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185 Quercetin glycosides were the only type of flavonols detected in red-fleshed

186 apples tested in this study. For both varieties, contents were significantly higher in the

187 peel than in the flesh. Bagging led to significantly reduced levels in the peels from

188 both varieties as well as in the flesh from ‘No.1 Hongxun’. By contrast, the contents

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189 of quercetin glycosides were enhanced in the flesh of ‘Xiahongrou’ apples that were

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190 bagged. Once the bags were removed, those contents were significantly improved in

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191 the peel, i.e., by 2.3 times and 1.8 times for ‘Xiahongrou’ and ‘No.1 Hongxun’,

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192 respectively, when compared with the control on Day 15. For flesh samples, bag

193 removal tended to decrease the levels of flavonols measured in ‘Xiahongrou’ but not

194
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in ‘No.1 Hongxun’. Feng et al. (2014) [18] have reported that debagging can
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195 enhanced the contents of flavonols in both the peel and flesh of ‘Jonagold’ apple.

196 Therefore, this contrast in results suggests that the accumulation of flavonols differs
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197 among apple varieties.

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198 We quantified two types of anthocyanins – cyanidin-3-galatoside and

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199 cyanidin-3-glucoside – and found that bagging treatment significantly reduced their
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200 contents in the peel and flesh of both varieties, although only small amounts were
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201 detected in our samples (Figure 2). After those bags were removed, anthocyanin

202 contents were increased in both varieties, with that response being weaker in the flesh,

203 especially for ‘No.1 Hongxun’. On Day 9, anthocyanin levels in ‘Xiahongrou’ flesh

204 were similar between bagged and control fruits, whereas the amount measured in the

205 ‘No.1 Hongxun’ flesh was only 10% of that detected in the control. This may have

206 been caused by a degradation in anthocyanin compounds within ‘No.1 Hongxun’

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207 during the maturation process.

208 Except for flesh samples from ‘Xiahongrou’, contents of total phenolics and

209 flavonoids decreased significantly in the flesh and especially in the peel of both

210 varieties in response to bagging. On Day 15, peels contained much higher levels when

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211 compared with the control. After the bags were removed, contents rose significantly in

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212 the flesh of ‘No.1 Hongxun’ but were not significantly changed in the flesh of

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213 ‘Xiahongrou’. On the other hand, tendencies of some indexes between bagged and

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214 control fruit were not the same during the experiment. Similar results were also

215 observed in the previous research on commercial white-fleshed apples that had been

216
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bagged [12,17,18]. According to Bizjak et al. (2013) [25], fruit development and
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217 maturity could significantly affect the phenolic composition of apples.

218 This inconsistency might be explained by delaying the maturation of bagged fruit.
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219 Chen et al. (2012) [24] have reported that bagging the fruit can significantly
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220 reduce contents of most phenolics, except chlorogenic acid, in apple peels.
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221 Furthermore, they have shown that anthocyanins are the most sensitive to such
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222 treatment, followed by flavonols. We found similar results here, with bagging being
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223 linked to a decline in contents of anthocyanins and flavonols in the peel from both

224 varieties. Decreases were also found for procyanidin B1, catechin, procyanidin B2,

225 and phloridzin in ‘Xiahongrou’ and for catechin in ‘No.1 Hongxun’. Sun et al. (2014)

226 [26] have shown that, when fruits are re-exposed to sunlight after having been bagged,

227 their contents of most phenolic compounds are markedly increased in the peel. We

228 obtained similar results here, with contents of individual phenolic compounds

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229 increasing significantly in the peel of both of our red-fleshed varieties after bag

230 removal.

231 Although fewer detailed quantitative analyses of phenolic compounds have been

232 conducted with apple flesh, Feng et al. (2014) [18] have demonstrated that, in samples

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233 of that tissue type from the commercial ‘Jonagold’ apple, bagging treatment decreases

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234 the levels of most phenolic compounds. After bags are removed, most of those

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235 contents increase slightly, reaching levels similar to those of the control. We found

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236 that the effect of such treatment on phenolic compounds in the flesh differed

237 significantly between genotypes. Similar to results by Feng et al. (2014) [18], our

238
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bagging treatment was linked to a decline in the amounts of most phenolic compounds
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239 in ‘No.1 Hongxun’ flesh, followed by a recovery once the bags were removed.

240 However, for ‘Xiahongrou’, contents of chlorogenic acid, procyanidin B1, catechin,
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241 and flavonols in the flesh were enhanced when fruits were bagged but were slightly
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242 decreased when the bags were removed. This difference in response to bagging and
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243 subsequent re-exposure to sunlight suggested that photonic regulation of phenolic

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244 compounds in the flesh might be genotype-dependent. By contrast, the responses of

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245 anthocyanins to bagging treatment were similar between the two varieties, indicating

246 that regulation of their synthesis by sunlight might be genotype-independent.

247 3.2. Effects of bagging on transcript levels for genes involved in anthocyanin

248 biosynthesis

249 The mRNA transcripts for genes involved in anthocyanin biosynthesis were

250 investigated via real-time PCR. Levels were significantly different between the peel

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251 and flesh from our two varieties. In the peel on Day 0 (Figure 3), transcripts for most

252 genes were much less abundant in bagged fruits than in the non-bagged control. After

253 bag removal, all genes were markedly up-regulated in the peels of both varieties, with

254 transcript levels peaking for nearly all of them within 9 d. Relative to values recorded

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255 on Day 0, maximum transcript levels for PAL, CHS, F3H, DFR, ANS, UFGT, FLS,

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256 LAR, ANR, MYB10, bHLH3, and bHLH33 were 76.6-, 29.4-, 8.3-, 9.5-, 28.1-, 13.3-,

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257 3771.0-, 5.3-, 8.6-, 10.1-, 2.2-, and 3.2-fold higher in ‘Xiahongrou’ and 19.7-, 97.4-,

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258 18.6-, 34.5-, 260.0-, 118.0-, 323.0-, 6.8-, 3.1-, 103.0-, 8.2-, and 17.4-fold higher in

259 ‘No.1 Hongxun’, respectively. These results with peel samples suggested that the

260
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genes most responsive to sunlight are PAL, CHS, ANS, and FLS in ‘Xiahongrou’ and
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261 CHS, ANS, UFGT, FLS, and MYB10 in ‘No.1 Hongxun’. Our findings from this

262 expression analysis also paralleled the significant increases in contents of

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263 anthocyanins and flavonols measured when peels were re-exposed to sunlight after the
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264 bags were removed.

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265 In flesh samples (Figure 4), transcript levels for genes involved in anthocyanin
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266 biosynthesis differed significantly between varieties. On Day 0, expressions for most
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267 genes were much lower in bagged fruits of ‘Xiahongrou’ than in the control. After bag

268 removal, however, transcript levels for most of them were increased to levels similar

269 to those detected in non-bagged fruits. However, for ‘No.1 Hongxun’, transcript levels

270 for F3H, DFR, FLS, LAR, ANR, bHLH3, and bHLH33 were similar to or even higher

271 in bagged fruits than in the control, suggesting that those genes were not sensitive to

272 the bagging treatment. After bag removal, transcript levels for all genes except PAL,

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273 CHS, ANS, UFGT, and MYB10 either decreased or remained unchanged when

274 compared with the control. Relative to levels on Day 0, transcripts of PAL, CHS, F3H,

275 DFR, ANS, UFGT, FLS, LAR, ANR, MYB10, bHLH3, and bHLH33 were increased by

276 7.5-, 3.2-, 9.0-, 2.9-, 5.5-, 3.8-, 313.0-, 1.6-, 9.7-, 19.6-, 1.5-, and 2.6-fold in

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277 ‘Xiahongrou’ and by 3.9-, 3.6-, 0.5-, 1.2-, 7.5-, 29.4-, 0.8-, 0.5-, 0.6-, 8.6-, 0.7-, and

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278 0.8-fold in ‘No.1 Hongxun’, respectively. These results suggested that PAL, F3H, FLS,

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279 ANR, and MYB10 in ‘Xiahongrou’ and ANS, UFGT, and MYB10 in ‘No.1 Hongxun’

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280 are the most responsive to sunlight re-exposure in the flesh of our two tested varieties.

281 The stronger upregulation by most genes in ‘Xiahongrou’ flesh reflected the

282
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difference in how anthocyanin contents were affected in both varieties.
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283 In apple fruits, PAL, CHS, F3H, DFR, ANS, and UFGT are the primary genes

284 involved in the anthocyanin biosynthetic pathway. Liu et al. (2013) [27] have shown
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285 that, during the ripening of non-red apples, UFGT may be the most important factor in
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286 anthocyanin biosynthesis, based on their measurements of activity by related enzymes.

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287 Wang et al. (2013) [12] have shown that DFR, ANS, and UFGT are the main genes
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288 that determine the synthesis of anthocyanins in ‘Granny Smith’ apple peels.
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289 Furthermore, Feng et al. (2014) [18] have indicated that debagging treatment leads to

290 the upregulation of expression by MYB10 and seven structural genes involved in

291 anthocyanin biosynthesis. Thus, expression by these genes appears to vary among

292 apple varieties. Similar results were found with our peel samples, in which

293 transcription of all genes involved in anthocyanin biosynthesis was markedly

294 up-regulated in both varieties. These results coincide with those from previous

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295 research on anthocyanin biosynthesis in the peel of commercial white-fleshed apples

296 that have been bagged [12,18,27]. However, we are not aware of any investigation of

297 how fruit bagging affects the expression of such genes in apple flesh. Using

298 blood-flesh peaches, Jiao et al. (2014) [28] have shown that, after bag removal,

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299 transcription levels for most anthocyanin biosynthetic genes in the mesocarp are

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300 markedly enhanced. That outcome is similar to what we observed here, with the

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301 exception of F3H in flesh samples from ‘No.1 Hongxun’. Therefore, we might

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302 conclude that a single gene is not responsible for anthocyanin accumulation but,

303 instead, coordinated action by many genes is necessary. Such a hypothesis has also

304
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been proven from apple experiments by Honda et al. (2002) [29].
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305 Furthermore, the FLS, LAR and ANR genes are correlated with the synthesis of

306 flavonols and flavanols [12]. Takos et al. (2006) [30] have shown that fruit bagging
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307 results in downregulation of the anthocyanin and flavonol pathway in apple peel,
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308 although transcripts of LAR and ANR are less affected by that treatment. We noted that
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309 transcripts of FLS were significantly enhanced in the peel after bag removal while
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310 those of ANR and LAR were only slightly elevated. This response was associated with
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311 changes seen in the levels of flavonols and flavanols. Our expression analysis with

312 flesh samples produced some unexpected results. For example, the increases in

313 transcripts for FLS, LAR, and ANR were more obvious in ‘Xiahongrou’ after bag

314 removal. However, changes in flavonols and flavanols contents were more significant

315 in ‘No.1 Hongxun’. Henry-Kirk et al. (2012) [31] have also reported discrepancies

316 between flavanol contents and expression by LAR and ANR in apple, which might be

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317 explained by spontaneous chemical degradation or an absence of substrate. Further

318 investigation is needed.

319 Many anthocyanin biosynthetic genes in apples are tightly regulated by

320 MYB-bHLH-WD40 TFs [12]. The behavior of MYB10 suggests that it is responsible

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321 for the red colour of apple fruit and those genes for anthocyanin production are

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322 regulated by that TF [10]. Furthermore, the variation in structure within its promoter

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323 region is responsible for the difference in MYB10 expression between white- and

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324 red-fleshed apples [7]. Investigation of bHLH proteins has revealed a close interaction

325 between MYBs and bHLHs [13]. In the present study, nearly all transcripts for

326
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MYB10, bHLH3, and bHLH33 from both varieties were less abundant in bagged fruit
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327 than in the control. Following bag removal, expression by most of those TFs was

328 enhanced, except for bHLH3 and bHLH33 in ‘No.1 Hongxun’. Nevertheless,
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329 expression by MYB10 was markedly higher in both peel and flesh after bag removal
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330 when compared with bHLH3 and bHLH33. Therefore these results confirm previous
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331 findings that it is the MYB component from the proposed regulatory complex of
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332 MYB-bHLH-WD40 that is primarily responsible for the extent of anthocyanin

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333 accumulation [10]. Likewise, our study results suggested that MYB10 is a key TF in

334 the anthocyanin pathway of red-fleshed apples, particularly in the flesh. This then

335 implies that the molecular mechanism for anthocyanin accumulation in the flesh after

336 bag removal is a consequence of light-induced coordinated expression by MYB10 and

337 the anthocyanin biosynthetic genes found in red-fleshed apples.

338 Studies elucidating the transcriptional regulation of anthocyanin biosynthesis in

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339 red-fleshed apples have advanced over the past decades [7,10,15,16]. However, the

340 effects of environmental controls on the regulatory patterns have not been fully

341 understood. The present study focused on the explanation of how environmental

342 factors influence the gene expression and the results showed that the expressions of

t
343 anthocyanin synthetic genes and TFs were affected in the two red-fleshed varieties

ip
344 when exposed to light. Our data also suggested that light was important for the

cr
345 development of flesh colour. Furthermore, MYB10 appeared to be the primary

us
346 determinant of fruit pigmentation in response to light. The expression level of MYB10

347 in the flesh was found to be higher in ‘Xiahongrou’ than that in ‘No.1 Hongxun’,

348
an
which coincided with the accumulation level of anthocyanin after the bag removal.
M
349 The findings indicated that different levels of anthocyanin accumulation between

350 varieties were due to different expression levels of light-induced TFs and the
d

351 anthocyanin biosynthetic genes, and were determined by the genetic background.
e

352 In this study, we examined the effect that fruit bagging has on levels of phenolic
pt

353 compounds in red-fleshed apples, and also monitored the expression of important
ce

354 anthocyanin biosynthetic and regulatory genes. The changes of phenolic compounds
Ac

355 and gene expression in peels found here coincided with reports from previous work

356 with commercial white-fleshed apples [12,18,24,26,27]. For our flesh samples,

357 bagging led to a decrease in anthocyanin contents, which were then partially

358 recovered after those bags were removed. Expression analysis revealed that

359 transcription was enhanced for most anthocyanin biosynthetic genes in the flesh,

360 except for F3H in ‘No.1 Hongxun’. The relative intensities of PAL, F3H in

17

Page 17 of 35
361 ‘Xiahongrou’ and ANS, UFGT in ‘No.1 Hongxun’ were the most responsive to

362 sunlight re-exposure in the flesh of the two varieties. Among TFs, the expressions of

363 MYB10 from both varieties were enhanced. We also determined that MYB10 is the key

364 TF in the anthocyanin pathway of red-fleshed apples, particularly in the flesh.

t
365 Counter-intuitively, the increases in FLS, LAR, and ANR expression were not

ip
366 associated with changes in the contents of flavonols and flavanols in flesh samples.

cr
367 We believe that these findings will improve our understanding of the molecular

us
368 mechanism for anthocyanin accumulation in red-fleshed apples.

369

370 Acknowledgements
an
M
371 This work was supported by the earmarked fund for the China Agriculture Research

372 System (CARS-28). The authors are grateful to Priscilla Licht for help in revising our
d

373 English composition.

e

374
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375
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Page 18 of 35
375 References

376 [1]. Carbone K, Giannini B, Picchi V, Lo Scalzo R, Cecchini F. Phenolic composition

377 and free radical scavenging activity of different apple varieties in relation to the

378 cultivar, tissue type and storage. Food Chem. 2011; 127: 493−500.

t
379 [2]. Kevers C, Pincemail J, Tabart J, Defraigne JO, Dommes J. Influence of cultivar,

ip
380 harvest time, storage conditions, and peeling on the antioxidant capacity and phenolic

cr
381 and ascorbic acid contents of apples and pears. J. Agric. Food Chem. 2011; 59:

us
382 6165−6171.

383 [3]. Zhang XJ, Wang LX, Liu YL, Chen XX, Yang YZ, Zhao ZY. Differential gene

384
an
expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit
M
385 skin coloration. S. Afr. J. Bot. 2013; 88: 125−131.

386 [4]. Würdig J, Flachowsky H, Höfer M, Peil A, Eldin Ali MAMS, Hanke MV.
d

387 Phenotypic and genetic analysis of the German Malus germplasm collection in terms
e

388 of type 1 and type 2 red-fleshed apples. Gene 2014; 544: 198−207.
pt

389 [5]. van Nocker S, Berry G, Najdowski J, Michelutti R, Luffman M, Forsline P,

ce

390 Alsmairat N, Beaudry R, Nair MG, Ordidge M. Genetic diversity of red-fleshed apple
Ac

391 (Malus). Euphytica 2012; 185: 281−293.

392 [6]. Yan G, Long H, Song W, Chen R. Genetic polymorphism of Malus sieversii

393 populations in Xinjiang, China. Genet. Resour. Crop Evol. 2008; 55: 171−181.

394 [7]. Espley RV, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill J,

395 Schouten HJ, Gardiner SE, Hellens RP, Allan AC. Multiple repeats of a promoter

396 segment causes transcription factor autoregulation in red apples. Plant Cell 2009; 21:

19

Page 19 of 35
397 168−183.

398 [8]. Xu W, Peng H, Yang T, Whitaker B, Huang L, Sun J, Chen P. Effect of calcium on

399 strawberry fruit flavonoid pathway gene expression and anthocyanin

400 accumulation. Plant Physiol. Biochem. 2014; 82: 289−298.

t
401 [9]. Feng F, Li M, Ma F, Cheng L. Phenylpropanoid metabolites and expression of

ip
402 key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in

cr
403 response to sun exposure. Plant Physiol. Biochem. 2013; 69: 54−61.

us
404 [10]. Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC.

405 Red colouration in apple fruit is due to the activity of the MYB transcription factor,

406 MdMYB10. Plant J. 2007; 49: 414−427.

an
M
407 [11]. Petroni K, Tonelli C. Recent advances on the regulation of anthocyanin synthesis

408 in reproductive organs. Plant Sci. 2011; 181: 219−229.

d

409 [12]. Wang L, Zhang X, Liu Y, Shi X, Wang Y, Zhang C, Zhao Z. The effect of fruit
e

410 bagging on the color, phenolic compounds and expression of the anthocyanin
pt

411 biosynthetic and regulatory genes on the ‘Granny Smith’ apples. Eur. Food Res.
ce

412 Technol. 2013; 237: 875−885.

Ac

413 [13]. Peng T, Moriguchi T. The molecular network regulating the coloration in

414 apple. Sci. Hortic. 2013; 163: 1−9.

415 [14]. Li X, Uddin MR, Park WT, Kim YB, Seo JM, Kim SJ, Nou IS, Lee J, Kim HR,

416 Park SU. Accumulation of anthocyanin and related genes expression during the

417 development of cabbage seedlings. Process Biochem. 2014; 49: 1084−1091.

418 [15]. Chagné D, Lin-Wang K, Espley RV, Volz RK, How NM, Rouse S, Brendolise C,

20

Page 20 of 35
419 Carlisle CM, Kumar S, Silva ND, Micheletti D, McGhie T, Crowhurst RN, Storey RD,

420 Velasco R, Hellens RP, Gardiner SE, Allan AC. An ancient duplication of apple MYB

421 transcription factors is responsible for novel red fruit-flesh phenotypes. Plant Physiol.

422 2013; 161: 225−239.

t
423 [16]. Umemura H, Otagaki S, Wada M, Kondo S, Matsumoto S. Expression and

ip
424 functional analysis of a novel MYB gene, MdMYB110a_JP, responsible for red flesh,

cr
425 not skin color in apple fruit. Planta 2013; 238: 65−76.

us
426 [17]. Liu Y, Zhang X, Zhao Z. Effects of fruit bagging on anthocyanins, sugars,

427 organic acids, and color properties of ‘Granny Smith’ and ‘Golden Delicious’ during

428
an
fruit maturation. Eur. Food Res. Technol. 2013; 236: 329−339.
M
429 [18]. Feng F, Li M, Ma F, Cheng L. The effects of bagging and debagging on external

430 fruit quality, metabolites, and the expression of anthocyanin biosynthetic genes in
d

431 ‘Jonagold’ apple (Malus domestica Borkh.). Sci. Hortic. 2014; 165: 123−131.
e

432 [19]. Zhang Y, Li P, Cheng L. Developmental changes of carbohydrates, organic acids,

pt

433 amino acids, and phenolic compounds in ‘Honeycrisp’ apple flesh. Food Chem. 2010;
ce

434 123: 1013−1018.

Ac

435 [20]. Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA

436 from pine trees. Plant Mol. Biol. Rep. 1993; 11: 113−116.

437 [21]. Telias A, Lin-Wang K, Stevenson DE, Cooney JM, Hellens RP, Allan AC,

438 Hoover EE, Bradeen JM. Apple skin patterning is associated with differential

439 expression of MYB10. BMC Plant Biol. 2011; 11: 93.

440 [22]. Li H, Flachowsky H, Fischer TC, Hanke MV, Forkmann G, Treutter D, Schwab

21

Page 21 of 35
441 W, Hoffmann T, Szankowski I. Maize Lc transcription factor enhances biosynthesis of

442 anthocyanins, distinct proanthocyanidins and phenylpropanoids in apple (Malus

443 domestica Borkh.). Planta 2007; 226: 1243−1254.

444 [23]. Awad MA, Wagenmakers PS, de Jager A. Effects of light on flavonoid and

t
445 chlorogenic acid levels in the skin of ‘Jonagold’ apples. Sci. Hortic. 2001; 88:

ip
446 289−298.

cr
447 [24]. Chen CS, Zhang D, Wang YQ, Li PM, Ma FW. Effects of fruit bagging on the

us
448 contents of phenolic compounds in the peel and flesh of ‘Golden Delicious’, ‘Red

449 Delicious’, and ‘Royal Gala’ apples. Sci. Hortic. 2012; 142: 68−73.

450
an
[25]. Bizjak J, Mikulic-Petkovsek M, Stampar F, Veberic R. Changes in primary
M
451 metabolites and polyphenols in the peel of “Braeburn” apples (Malus domestica

452 Borkh.) during advanced maturation. J Agri Food Chem. 2013; 61: 10283−10292.
d

453 [26]. Sun S, Xin L, Gao H, Wang J, Li P. Response of phenolic compounds in ‘Golden
e

454 Delicious’ and ‘Red Delicious’ apples peel to fruit bagging and subsequent sunlight
pt

455 re-exposure. Sci. Hortic. 2014; 168: 161−167.

ce

456 [27]. Liu Y, Che F, Wang L, Meng R, Zhang X, Zhao Z. Fruit coloration and
Ac

457 anthocyanin biosynthesis after bag removal in non-red and red apples (Malus ×

458 domestica Borkh.). Molecules 2013; 18: 1549−1563.

459 [28]. Jiao Y, Ma RJ, Shen ZJ, Yan J, Yu ML. Gene regulation of anthocyanin

460 biosynthesis in two blood-flesh peach (Prunus persica (L.) Batsch) cultivars during

461 fruit development. J Zhejiang Univ-Sci. B 2014; 15: 809−819.

462 [29]. Honda C, Kotoda N, Wada M, Kondo S, Kobayashi S, Soejima J, Zhang Z,

22

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463 Tsuda T, Moriguchi T. Anthocyanin biosynthetic genes are coordinately expressed

464 during red coloration in apple skin. Plant Physiol. Biochem. 2002; 40: 955−962.

465 [30]. Takos AM, Robinson SP, Walker AR. Transcriptional regulation of the flavonoid

466 pathway in the skin of dark-grown 'Cripps' Red' apples in response to sunlight. J.

t
467 Horticult. Sci. Biotechnol. 2006; 81: 735−744.

ip
468 [31]. Henry-Kirk RA, McGhie TK, Andre CM, Hellens RP, Allan AC. Transcriptional

cr
469 analysis of apple fruit proanthocyanidin biosynthesis. J. Exp. Bot. 2012; 63:

us
470 5437−5450.

471
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471 Figure Captions:

472 Figure 1 Scheme of the apple phenylpropanoid pathway, showing structural genes

473 involved in flavonoid biosynthesis. PAL, phenylalanine ammonia lyase; CHS,

474 chalcone synthase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase;

t
475 ANS, anthocyanidin synthase; UFGT, UDP-glucose flavonoid 3-O-glucosyl

ip
476 transferase; FLS, flavonol synthase; LAR, leucoanthocyandin reductase; ANR,

cr
477 anthocyanidin reductase.

us
478 Figure 2 Bagging-treated and control (non-bagged) fruit from two red-fleshed apple

479 varieties, shown at Day 0.

480
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Figure 3 Effects of bagging and subsequent sunlight re-exposure on transcript levels
M
481 for key genes involved in anthocyanin synthesis in peel from red-fleshed apples at 0, 3,

482 6, 9, and 15 d after bag removal. Actin served as internal control for real-time PCR
d

483 analysis. Black column, bagged fruit; gray column, non-bagged control. Each point is
e

484 mean ± SD (n = 3).

pt

485 Figure 4 Effects of bagging and subsequent sunlight re-exposure on transcript levels
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486 for key genes involved in anthocyanin synthesis in flesh from red-fleshed apples at 0,
Ac

487 3, 6, 9, and 15 d after bag removal. Actin served as internal control for real-time PCR

488 analysis. Black column, bagged fruit; gray column, non-bagged control. Each point is

489 mean ± SD (n = 3).

24

Page 24 of 35
 cr
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Table 1
Effects of fruit bagging and subsequent sunlight re-exposure on concentrations of phenolic compounds in peel samples from red-fleshed apples

M
at 0, 3, 6, 9, and 15 d after bag removal. Note: Values are means ± SD values of three replicates. Data were subjected to a t-test. Asterisks
indicate significant differences between the bagged and the control (n = 3, P < 0.05).
‘Xiahongrou’ ‘No.1 Hongxun’

ed
Day after bag removal 0 3 6 9 15 0 3 6 9 15

Chlorogenic acid Bagged 47.2±1.2 53.3±1.1* 51.8±0.8* 47.2±1.3* 47.4±0.8 123.3±2.9 116.2±5.5g* 156.8±5.6* 198.2±2.5* 183.4±4.1*

Control 48.3±1.3 45.3±2.5 38.5±0.9 36.8±0.6 48.5±1.5 122.3±2.4 127.1±0.5 130.2±5.4 145.7±2.4 148.9±3.2
pt
Phloridzin Bagged 166.6±5.6* 216.7±9.9 213.3±17.6 230.7±3.1 254.6±4.2* 69.0±3.9 68.5±2.0* 108.6±6.8* 230.1±16.9* 106.5±5.3*
ce
Control 215.8±6.6 223.8±7.1 228.8±13.4 223.8±4.5 237.5±6.3 61.6±3.8 93.1±1.5 73.8±2.8 81.2±6.4 86.7±9.4

Procyanidin B1 Bagged 192.4±10.5* 237.8±15.4 250.4±10.2 275.8±11.8 251.5±3.6 195.3±6.8 174.6±4.4* 227.2±8.0* 236.5±5.6* 240.9±10.5*
Ac

Control 231.5±5.7 232.8±5.2 245.2±11.7 272.6±11.6 265.1±20.9 200.9±6.4 226.0±5.3 207.8±9.1 206.2±15.8 221.0±7.4

Catechin Bagged 133.4±4.8* 178.2±7.0* 186.2±6.6* 207.1±2.6* 200.6±2.3* 101.7±5.1* 93.9±3.3* 133.6±2.2* 116.6±4.4 96.1±3.6*

Control 169.5±14.5 163.4±0.8 168.0±5.9 184.4±4.0 173.3±1.7 148.9±4.4 133.0±1.3 149.3±4.9 111.5±4.5 81.8±5.8

Procyanidin B2 Bagged 203.7±12.4* 228.5±6.7 235.7±13.4 253.7±3.8* 248.7±0.7* 114.7±4.9 98.8±3.3* 137.8±4.3* 132.9±3.8* 131.8±2.5*

Control 243.4±13.2 238.9±3.8 247.9±5.1 274.7±2.3 269.2±3.3 115.1±1.7 121.0±1.1 126.3±7.1 105.0±3.2 111.1±10.4

Epicatechin Bagged 91.4±1.3 104.8±3.7* 102.7±2.9* 101.4±6.0* 108.5±5.6 123.5±5.7 80.2±4.4* 196.2±3.2* 214.2±5.9* 155.6±6.5

Control 94.6±5.3 95.6±3.0 89.8±1.3 92.2±3.2 102.3±0.4 129.2±5.3 135.7±3.6 124.2±13.4 141.2±5.4 166.3±3.3

Sum of Flavanols Bagged 620.8±15.8* 749.3±32.0 775.0±29.9 838.0±21.9 809.3±2.1 535.2±22.2* 447.5±10.8* 694.8±15.5* 700.2±17.6* 624.4±19.3*

Control 738.9±34.0 730.7±7.7 750.8±20.4 823.9±5.8 809.9±16.8 594.1±17.2 615.6±8.2 607.5±33.4 563.9±23.5 580.2±16.0

Quercetin-3-galactoside Bagged 34.0±2.0* 157.6±12.8* 164.6±11.3* 253.0±6.8* 406.8±9.4* 99.4±5.2* 290.8±15.8* 426.3±20.9* 410.9±21.0* 404.2±24.0*

Page 25 of 35
 cr
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Control 95.5±5.6 123.7±1.6 82.9±5.1 100.2±2.8 158.8±3.2 190.9±13.3 203.6±4.4 183.1±16.9 186.1±5.3 222.0±14.2

Quercetin-3-rutinoside Bagged 0.8±0.2* 6.0±0.5* 5.4±0.7* 13.0±0.5* 18.2±0.4* 0.9±0.2* 3.1±0.1* 5.2±0.4* 4.4±0.3* 6.9±0.6*

Control 2.7±0.4 3.8±0.1 2.2±0.2 2.2±0.0 4.9±0.2 1.6±0.3 1.9±0.3 1.8±0.3 1.8±0.1 1.8±0.4

M
Quercetin-3-glucoside Bagged 16.4±0.9* 53.7±3.3* 57.8±5.2* 82.3±2.3* 129.9±3.7* 28.0±1.3* 58.6±3.1* 83.8±4.1* 74.8±3.6* 78.1±4.4*

Control 30.3±2.6 40.0±0.2 27.4±1.1 32.4±1.2 53.9±1.3 35.1±1.1 39.9±0.6 35.1±4.6 35.7±1.1 34.7±4.6

Quercetin-3-xyloside Bagged 13.6±0.3* 34.6±2.2* 39.2±3.5* 46.8±1.6* 70.8±1.7* 27.2±1.4* 43.2±2.6 60.5±3.6* 56.3±2.7* 43.0±2.1*

Control 22.0±2.4 29.0±0.2 22.2±1.6 25.8±1.6 38.6±1.2 37.4±1.5 39.8±0.4 35.6±4.4 34.0±0.8 33.5±4.8

ed
Quercetin-3-arabinoside Bagged 31.6±1.1* 76.6±5.4* 85.0±6.2* 102.3±2.5* 147.6±3.5* 59.5±3.3* 101.2±6.4* 137.4±7.7* 137.4±7.1* 119.4±6.0*

Control 44.2±2.8 57.9±1.3 44.4±2.6 49.8±2.1 74.6±1.5 80.3±5.0 83.3±1.3 72.8±4.7 72.6±1.9 76.9±4.4

Quercetin-3-rhamnoside Bagged 2.8±0.3 6.0±0.3* 7.0±0.6* 7.9±0.3* 10.2±0.3* 7.2±0.4* 9.7±0.7 12.3±0.8* 11.1±0.6* 7.3±0.3*
pt
Control 2.8±0.1 3.4±0.3 3.3±0.2 3.9±0.3 5.5±0.1 9.5±0.6 9.3±0.1 8.7±0.6 7.7±0.3 6.2±1.0

Sum of Flavonols Bagged 99.2±3.6* 334.4±23.9* 358.9±27.3* 505.4±13.8* 783.5±19.0* 222.3±11.4* 506.7±28.6* 725.4±37.4* 694.8±34.8* 658.8±37.1*
ce
Control 197.5±13.8 257.7±3.4 182.3±10.0 214.4±7.8 336.4±7.4 354.8±21.2 377.8±6.5 337.2±30.9 337.9±8.8 375.1±29.2

Cyanidin-3-galactoside Bagged 5.7±0.3* 97.1±10.8* 175.7±7.0* 341.7±12.3* 476.3±5.0* 6.9±0.3* 190.3±6.9* 286.0±11.2* 335.3±20.0* 376.0±19.5*

Control 190.2±5.0 180.2±8.8 226.0±5.7 258.7±3.2 245.8±1.6 336.2±12.2 362.4±6.3 369.3±6.1 261.0±6.4 255.6±8.4
Ac

Cyanidin-3-glucoside Bagged 0.2±0.0* 0.6±0.1 0.8±0.1 1.2±0.1* 1.5±0.2* 0.2±0.0* 0.7±0.1* 1.1±0.0 1.3±0.1* 1.5±0.1

Control 0.6±0.3 0.5±0.1 0.7±0.2 0.8±0.1 0.8±0.2 1.6±0.3 1.4±0.1 1.4±0.1 0.9±0.2 1.4±0.2

Sum of Anthocyanins Bagged 5.9±0.3* 97.7±10.7* 176.4±7.1* 342.9±12.2* 477.8±5.2* 7.1±0.2* 191.0±7.0* 287.1±11.2* 336.6±20.1* 377.5±19.6*

Control 190.7±5.3 180.7±8.9 226.7±5.8 259.5±3.3 246.7±1.8 337.7±12.2 363.8±6.4 370.7±6.1 261.9±6.5 257.0±8.6

Sum of individual flavonoids Bagged 725.9±14.9* 1181.4±65.2 1310.3±62.5* 1686.3±47.1* 2070.6±23.0* 764.5±32.6* 1145.2±26.0* 1707.2±62.0* 1731.6±68.8* 1660.8±37.3*

Control 1127.2±52.9 1169.1±4.0 1159.8±35.4 1297.8±12.2 1392.9±9.2 1286.6±47.5 1357.2±21.1 1315.3±68.8 1163.7±32.8 1212.3±52.4

Sum of individual phenolics Bagged 961.5±19.1* 1474.3±74.6 1601.5±78.6* 1992.0±51.0* 2400.0±27.2* 985.6±36.6* 1351.3±19.3* 2008.2±51.5* 2190.8±84.3* 1974.4±28.9*

Control 1412.8±61.3 1462.7±2.0 1451.4±49.3 1585.1±16.6 1706.6±4.3 1492.1±52.8 1603.6±21.9 1544.1±72.3 1411.1±29.1 1468.3±63.9

Page 26 of 35
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Table 2
Effects of fruit bagging and subsequent sunlight re-exposure on concentrations of phenolic compounds in flesh samples from red-fleshed apples

M
at 0, 3, 6, 9, and 15 d after bag removal. Note: Values are means ± SD values of three replicates. Data were subjected to a t-test. Asterisks
indicate significant differences between the bagged and the control (n = 3, P < 0.05).
‘Xiahongrou’ ‘No.1 Hongxun’

ed
Day after bag removal 0 3 6 9 15 0 3 6 9 15

Chlorogenic acid Bagged 55.9±1.7* 56.3±2.3* 46.5±1.1 49.2±0.1* 46.7±2.3 165.5±2.1 205.7±3.1* 233.3±4.7* 235.9±6.4* 204.5±13.9*

Control 50.2±1.8 50.1±3.5 47.4±1.8 42.0±1.2 44.0±1.5 159.2±2.6 161.8±1.6 167.7±1.4 171.6±2.0 182.7±0.6
pt
Phloridzin Bagged 22.2±0.4 20.3±2.5 17.6±0.7 14.8±1.2* 17.1±1.0* 16.2±2.9 21.6±0.8 25.8±3.5b* 36.0±4.1* 33.4±3.3*
ce
Control 19.9±3.3 20.0±1.8 18.9±1.3 19.6±1.5 22.6±2.7 13.6±2.1 18.8±3.2 17.3±4.3cd 14.7±1.1 21.5±0.9

Procyanidin B1 Bagged 73.7±4.1* 63.7±3.8* 69.1±3.3* 79.0±3.2* 74.1±2.5* 46.6±1.4* 47.2±1.7* 73.4±4.6* 61.5±3.4* 61.7±3.3*
Ac

Control 48.3±2.5 51.8±3.4 60.1±4.2 61.2±3.4 54.8±3.1 66.9±2.6 56.9±2.3 63.1±2.2 50.9±2.5 47.3±2.6

Catechin Bagged 38.5±1.7* 36.7±3.1 37.1±1.9 41.6±3.8 36.7±2.9 12.8±0.8* 15.5±1.1* 23.7±0.9* 19.3±0.2 19.1±2.8*

Control 30.4±3.2 32.3±3.0 39.5±3.6 39.0±1.8 34.7±4.0 35.7±3.2 29.6±1.3 38.0±6.0 20.7±0.7 14.4±3.0

Procyanidin B2 Bagged 63.8±2.7 59.6±1.0 62.9±2.3* 72.6±3.6* 63.4±2.2 11.7±0.8* 13.9±1.1* 25.0±0.5 21.5±0.7 18.6±2.9

Control 62.7±1.8 58.1±4.2 71.9±3.2 66.7±2.6 67.6±3.2 25.0±2.0 24.1±1.5 28.5±4.4 18.1±0.6 15.3±4.1d

Epicatechin Bagged 24.6±0.8 26.9±1.8 24.7±3.3 17.8±1.5* 14.3±1.9* 56.0±0.9 74.8±1.7* 102.2±1.9 109.7±3.1 102.3±4.6

Control 27.8±1.7 27.7±2.4 27.0±1.5 26.0±1.0 34.3±1.4 56.1±3.3 42.7±2.9 49.8±2.9 61.8±0.1 66.3±0.4

Sum of Flavanols Bagged 200.5±8.5* 186.9±6.4* 193.8±6.6 211.0±9.0* 188.5±8.7 127.1±2.4* 151.4±1.5 224.3±2.5* 212.0±6.6* 201.7±12.3*

Control 169.2±1.8 169.9±5.1 198.4±11.0 192.9±3.9 191.4±10.7 183.7±10.9 153.3±5.8 179.4±11.2 151.5±1.5 143.2±8.9

Quercetin-3-galactoside Bagged 3.7±0.2* 2.2±0.3 3.0±1.0* 2.0±0.1 1.9±0.5 1.0±0.3* 2.2±0.9 2.5±0.4* 2.2±0.9 2.9±1.0*

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Control 1.9±0.5 1.7±0.4 1.4±0.1 1.9±0.7 1.9±0.4 2.7±0.3 3.9±0.9 4.3±0.8 3.1±0.5 4.6±1.8

Quercetin-3-rutinoside Bagged 0.6±0.2 0.7±0.2 0.6±0.1 0.4±0.1 0.4±0.2 0.1±0.0* 0.2±0.1* 0.2±0.0* 0.3±0.0 0.5±0.0

Control 0.6±0.1 0.5±0.2 0.5±0.1 0.5±0.2 0.4±0.1 0.5±0.1 0.5±0.1 1.4±0.2 0.4±0.2 0.7±0.3

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Quercetin-3-glucoside Bagged 1.5±0.1* 1.3±0.3* 1.4±0.5 0.9±0.3 0.8±0.3 0.5±0.0* 0.8±0.2* 1.0±0.1* 0.9±0.1 1.1±0.2*

Control 1.0±0.2 0.7±0.2 0.9±0.1 0.8±0.2 0.9±0.1 2.3±0.6 1.9±0.2 2.1±0.7 1.2±0.1 2.0±0.4

Quercetin-3-xyloside Bagged 1.3±0.2* 0.9±0.2 0.9±0.5 1.1±0.6 0.7±0.3 0.4±0.1* 0.6±0.1* 0.6±0.2* 0.6±0.0* 0.7±0.2*

Control 0.5±0.1 0.6±0.1 1.2±0.2 0.8±0.3 0.8±0.2 1.6±0.5 1.4±0.4 1.9±0.4 1.1±0.1 1.4±0.3

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Quercetin-3-arabinoside Bagged 2.5±0.8* 1.9±0.5* 1.5±0.6 1.3±0.5 1.4±0.1 1.0±0.3* 1.2±0.1* 1.2±0.1* 1.2±0.1* 1.4±0.3*

Control 1.1±0.2 0.7±0.0 2.1±0.2 1.1±0.7 1.1±0.1 2.3±0.7 2.4±0.3 2.9±0.8 2.1±0.1 2.9±0.8

Quercetin-3-rhamnoside Bagged 0.6±0.2 0.5±0.2 0.4±0.0 0.4±0.2 0.4±0.1 0.3±0.0* 0.4±0.0* 0.5±0.0* 0.4±0.0* 0.4±0.1*
pt
Control 0.4±0.1 0.3±0.1 0.6±0.1 0.5±0.1 0.3±0.0 0.8±0.2 1.2±0.1 0.9±0.1 1.0±0.3 1.1±0.3

Sum of Flavonols Bagged 10.2±1.2* 7.44±0.8* 7.7±2.5 6.25±1.2 5.6±1.0 3.3±0.7* 5.3±1.3* 5.9±0.8* 5.5±1.0* 6.9±1.4*
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Control 5.5±0.9 4.5±1.1 6.7±0.4 5.5±1.0 5.3±0.6 10.1±1.8 11.4±1.0 13.4±2.6 8.8±0.5 12.7±3.3

Cyanidin-3-galactoside Bagged 6.9±0.9* 8.9±0.5* 14.2±0.5* 32.5±2.0* 29.5±1.7* 2.6±0.2* 8.4±0.8* 10.5±0.9* 15.8±0.8* 13.5±1.6*
Ac

Control 29.3±0.8 34.0±1.9 38.1±0.6 35.0±1.0 41.3±1.3 76.4±3.2 96.3±0.5 104.8±3.0 73.5±0.7 44.3±1.5

Cyanidin-3-glucoside Bagged 0.2±0.1b* 0.2±0.0b 0.3±0.0 0.3±0.1 0.3±0.1 0.2±0.0 0.2±0.0 0.2±0.1* 0.2±0.1 0.7±0.1

Control 0.4±0.0a 0.3±0.1ab 0.2±0.1 0.2±0.0 0.2±0.1 0.5±0.2 0.5±0.0 0.8±0.1 0.3±0.1 0.7±0.5

Sum of Anthocyanins Bagged 7.1±0.9* 9.1±0.5* 14.4±0.5* 32.9±1.9 29.8±1.7* 2.8±0.3* 8.7±0.8* 10.7±1.0* 16.0±0.8* 14.1±1.5*

Control 29.6±0.8 34.4±2.0 38.3±0.6 35.2±1.0 41.5±1.2 76.9±3.2 96.7±0.5 105.7±3.1 73.8±0.8 45.0±2.0

Sum of individual flavonoids Bagged 217.7±9.0 203.4±6.3 215.8±8.7* 250.1±9.6 223.9±10.0 133.3±2.9* 165.3±1.3* 240.9±3.7* 233.5±6.0 222.7±14.8

Control 204.3±2.4 208.8±4.9 243.4±11.8 233.6±4.0 238.2±12.5 270.7±15.2 261.4±6.3 298.5±13.0 234.1±0.8 200.9±3.9

Sum of individual phenolics Bagged 304.4±10.8* 288.3±10.5 287.1±9.6* 322.0±10.6 295.0±10.9 320.6±4.6* 398.5±1.2* 509.7±4.3 513.0±9.7* 469.5±25.0*

Control 283.0±2.3 287.1±6.4 317.6±14.8 303.1±6.4 312.8±15.9 451.7±16.9 449.6±9.4 492.5±9.9 426.1±2.6 412.9±5.3

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Black and white version of Fig. 2

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*Highlights (for review)

Highlights

1. The bagging effect on red-fleshed apples was evaluated for the first time.

2. The accumulation of anthocyanin in apple flesh is sensitive to light.

3. The molecular mechanism for red pigmentation in apple flesh was analyzed.

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*Graphical Abstract

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