Significant Reduction of the Expression of Peach (Prunus persica L

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Agricultural and Environmental Chemistry

Fruit Bagging with Opaque Paper Significantly Reduced the Expression of Peach (Prunus persica L. Batsch) Allergen-Encoding Genes Yingtao Ma, Xuejiao Zhao, Hongwei Ren, Hongxia Wu, Mingxin Guo, Yanzhao Zhang, Zhaojun He, Jianming Han, and Ruijian Tong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00207 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Journal of Agricultural and Food Chemistry

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Fruit Bagging with Opaque Paper Significantly Reduced

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the Expression of Peach (Prunus persica L. Batsch)

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Allergen-Encoding Genes

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Yingtao Ma,*† Xuejiao Zhao,† Hongwei Ren,‡

5

Yanzhao Zhang,†

Hongxia Wu,§ Mingxin Guo,

†

Zhaojun He,ǁ Jianming Han, † and Ruijiang Tong†

6 7

†

Life Science College, Luoyang Normal University, Luoyang, Henan 471934, China

8

‡

Institute of Fruit Science, Luoyang Academy of Agriculture and Forestry, Luoyang,

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Henan 471000, China

10

§

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Agricultural Sciences, Zhanjiang, Guangdong 524091, China

12

ǁ

13

Henan 471934, China

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*Author to whom correspondence should be addressed (Telephone: +86 18103889053;

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E-mail: [email protected])

South Subtropical Crops Research Institute, Chinese Academy of Tropical

College of Food and Pharmaceutical Sciences, Luoyang Normal University, Luoyang,

16 17 18 19 20

ACS Paragon Plus Environment


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ABSTRACT

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Freshly consumed peaches (Prunus persica L. Batsch) can cause allergic reactions in

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the worldwide population because of the presence of four classes of allergens (Pru p 1,

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Pru p 2, Pru p 3, and Pru p 4). Fruit bagging has been widely practiced in peach

25

cultivation to improve fruit quality; however, its effect on the expression of peach

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allergen-encoding genes remains unknown. In this study, the influence of fruit

27

bagging with opaque paper bags on the major peach allergen-encoding genes,

28

including Pru p 1.01, Pru p 1.06B, Pru p 2.01B, Pru p 2.02, Pru p 3.01, Pru p 4.01,

29

and Pru p 4.02, were measured by means of real-time PCR. A significant reduction in

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transcript accumulation was observed for all of the selected genes in the epicarp of the

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bagged peach fruits, while a slight increase was observed in the mesocarp for these

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genes, with the two exceptions of Pru p 2.02 and Pru p 3.01. For most of these genes,

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much higher transcripts were determined in the epicarp than in the mesocarp. Taken

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together, a significant reduction in the transcription rate of the allergen-encoding

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genes in the whole peach fruit was achieved by shading with opaque paper bags.

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According to these data, modifications in growing practices of peach may help to

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obtain fruits with lower levels of allergens, and thus contribute to reducing potential

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allergenic risks in consumers.

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KEYWORDS

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Peach; Allergen; Fruit bagging; Gene expression; Real-time PCR


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INTRODUCTION

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Fruits are widely consumed all around the world and considered to be an important

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component of a healthy diet. However, fruits can also pose a major threat to the health

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of those who are allergic to them.1 An increasing prevalence of fruit allergies has been

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reported in recent decades.2-3 The majority of allergens in Rosaceae fruit have been

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reported to belong to four families: pathogenesis-related protein 10 (PR-10 protein,

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birch allergen Bet v 1 homologues), thaumatin-like proteins (TLP, PR-5 proteins),

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non-specific lipid transfer proteins (nsLTPs, PR-14 proteins), and profilins.4

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Originally from China, the peach (Prunus persica) is one of the most frequently

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reported allergenic fruits.5-6 Four allergen families, denoted as Pru p 1 (PR-10),7 Pru p

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2 (TLP),8 Pru p 3(LTP),9 and Pru p 4 (profilin),10 have been identified in peaches.

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Among Pru p 1, two members (Pru p 1.01 and Pru p 1.06B) have been found to be the

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most abundant in peaches. Among Pru p 2, Pru p 2.01B and Pru p 2.02 have been

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demonstrated to be the predominant members. Pru p 3.01 is the only Pru p 3 family

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member that accumulates in the fruit, while the others are almost undetectable. The

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two members of Pru p 4, Pru p 4.01 and Pru p 4.02, show similar transcript abundance

57

in the fruit.11

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For patients with fruit allergies, the general avoidance of fruit has a negative

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effect on their health. Knowledge of environmental and endogenous factors affecting

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the allergenic potential of fruits would provide important information to fruit breeders,

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growers, and consumers for the selection of hypoallergenic genotypes, the adoption of

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agronomical practices decreasing allergenic potential, and the consumption of fruit



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with reduced amounts of allergens. Gene expression data are now available for apples,

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and some authors12-13 have demonstrated that fruit storage conditions significantly

65

affect the transcription of apple allergen Mal d 1. Shading, elevation, and water stress

66

can also influence the expression of apple allergens.14-15 The influence of ethylene on

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the expression of genes encoding allergens in apples has also been investigated, and

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significant increases in the transcript abundance were found after ethylene

69

treatment.16 Kiewning et al. found that the Mal d 1 content of apples was significantly

70

reduced by treatment with 1-methylcyclopropene (1-MCP) during storage.13 As for

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peaches, Botton et al. revealed that storage at low temperatures induces a decrease of

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Pru p 3 transcript accumulation in two cultivars, ‘Sentry’ and ‘Tardiva Zuliani’.17 The

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same group also demonstrated that a reduction of the transcription rate of most peach

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allergen-encoding genes could be achieved by enhancing intracanopy light radiation

75

and decreasing fruit load.18

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As a good agricultural practice (GAP), fruit bagging has been widely used in

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fruit cultivation to not only protect fruit against damage from pests, birds, diseases,

78

and mechanical scratches, but it also reduces pesticide residues.19 Lin et al. reported

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that pesticide residues of 'Cuiguan' and 'Hosui' pear fruits were significantly reduced

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by bagging treatment.20 In earlier studies, fruit bagging had been reported to improve

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the visual quality of fruits by promoting skin coloration and reducing blemishes. Jia et

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al. pointed out that when the 'Hakuho' peach was covered with orange paper bags it

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had a bright red skin, which accounted for the high visual quality.21 Huang et al.

84

reported that the attractive appearance of the red Chinese sand pear 'Meirensu' can be


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obtained by covering the fruit with light-impermeable bags at the early stage of fruit

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development.22 Fruit bagging is also an effective way to improve fruit color in

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apples.23 However, it is believed that bagging can also change the micro-environment

88

for fruit development and affect the internal quality of fruit. Jia et al. demonstrated

89

that bagging treatment caused earlier peach ripening as well as low anthocyanin and

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high lactone levels.21 Bagging treatment with light-impermeable, double-layer paper

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bags decreased most of the phenolic compound concentrations in apple fruit.24 Xu et

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al. reported that opaque paper bags (a black inner layer and a grey outer layer)

93

decreased the inner quality and total antioxidant capacity of loquat fruit.25 Wang et al.

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reported that bagging with opaque paper bags (black inner and brown outer paper)

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accelerated fruit maturity and reduced volatile contents, keracyanin, and

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quercetin-3-rutinoside contents in “Wanmi” peach.26 However, the effects of fruit

97

bagging on the expression of fruit allergen-encoding genes remains unclear. The

98

purpose of the present study was to investigate the effects of bagging with

99

light-impermeable paper bags on the expression levels of major peach

100

allergen-encoding genes. The experimental findings and potential practical

101

implications for common horticultural practices are discussed.

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MATERIALS AND METHODS

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Plant materials and bagging treatment

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Five-year-old P. persica cv. Zaochunhong trees growing at the experimental

105

orchard of Luoyang Academy of Agriculture and Forestry Science (Henan, China)



106

were used in this study. ‘Zaochunhong’ is an extremely early-maturing peach

107

cultivar with strong biotic and abiotic stress resistance, and its fruit is large,

108

white-fleshed, clingstone, and with a firm melting flesh texture that is suitable for

109

both fresh markets and processing. The research was carried out on peaches

110

harvested for two subsequent growing seasons (2016 and 2017). However, since the

111

results collected in 2016 were confirmed in 2017, at least in terms of general trends,

112

only the former are reported in the present study, while data concerning 2017 are

113

presented in Supplementary Figure S1.

114

Before the pit-hardening stage (April 29, 2016, i.e., 36 days after anthesis), 100

115

fruits (uniform in size without visible defects) of 10 homogeneous trees were

116

divided into two groups for bagging and non-bagging treatment. The selected fruits

117

were distributed on the southern or western sides of the trees and vertically in the

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up and middle position within 1.5 m ~ 3.0 m of the canopy, receiving mean relative

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light intensity of approximately 49.2%. Bagged fruits were on-tree covered with

120

double-layered paper bags (18.0 × 15.0 cm, the outer and inner layers were brown

121

paper and black waxed paper, respectively; waterproof and ~0.1% transmission of

122

sunlight; Laiyang, Shandong, China), whereas non-bagged fruits were kept as a

123

control under normal light.

124

Fruits were harvested (66 days after anthesis) at commercial maturity as

125

described by Kader and Mitchell,27 with mean flesh firmness, total soluble solids

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(TSS), and titratable acidity (TA) of control fruits of 42.80 N, 11.75 %, and 0.193 %,

127

respectively. To avoid their exposure to light before the quality analysis, the bagged


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fruits were collected without removing the bags. Uniform fruits free of visible

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defects were screened and then sampled on the day of collection without any

130

postharvest storage treatment.

131

Fruit physicochemical quality evaluation and tissue sampling

132

The fruit weight and dimension, presented as longitudinal diameter (LD) and

133

horizontal diameter (HD), were measured immediately after harvest. Fruit firmness

134

measurements were made on two equidistant points on the equatorial region of each

135

fruit after the removal of a 1-mm thick skin slice with a TA-XT2i texture analyzer

136

(Stable Micro Systems®, UK) equipped with a 7.9-mm plunger, and data were

137

expressed in Newtons (N). TSS were measured from both ends of each fruit, using a

138

N20 digital hand-held refractometer (Atago, Japan) according to the methods

139

described by Zhang et al.28 TSS data were expressed in °Brix (concentration of

140

sucrose w/w). TA was determined with 25 g of pulp homogenized with 75 mL

141

distilled water and measured using a DL15 titrator (Mettler-Toledo, USA), and the

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results were expressed as % of malic acid.

143

The epicarp, including the outer epidermis and a few layers of hypodermal cells,

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and mesocarp, a 1-cm-thick region of the mesocarp closest to the epicarp, were

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excised separately, immediately frozen in liquid nitrogen, and then kept at -80°C for

146

further experiments.

147 148

Total protein was extracted from the fruit epicarp and mesocarp, respectively, according to the methods elaborated previously by Gao et al.29 The fruit sample (1 g)



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was ground and homogenized in Coca’s solution (0.1 M Na2CO3, 0.1 M NaHCO3,

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0.1 M NaCl, 2 mM EDTA-Na2, and 20 mM sodium diethyidithiocarbamate

151

trihydrate) (5 mL). The mixture was stirred continuously at 4.0°C for 1 h and then

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centrifuged at 12,000 rpm at 4°C for 1 h. The absorbance of the supernatant was

153

measured using a DU800 spectrophotometer (Beckman, Fullerton, CA, USA) at

154

595 nm. The protein content was calculated with a Bradford Protein Assay Kit

155

(Takara, Japan), in specific accordance with the instructions for use.

156

RNA Extraction and cDNA Synthesis

157

Total

RNA

was

isolated

from

pooled

samples

158

cetyltrimethyl-ammonium bromide (CTAB) method.30 For each sample, 1 g of

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dissociative fruit tissues were extracted in 10 ml of extraction buffer.

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Contaminated genomic DNA was removed by RNase-free DNase I (TaKaRa,

161

Japan) treatment. The purified total RNA was quantified with a ND-3300

162

spectrophotometer (Thermo Fisher, USA ) at wavelengths of 260 and 280 nm. The

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integrity of total RNA was verified by running samples on 1.2% denaturing

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agarose gels (Qiagen, RNeasy Mini). First-strand cDNA was synthesized using 1.0

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g of treated RNA for each sample with AMV Reverse Transcriptase XL (TaKaRa,

166

Japan) and oligo (dT) as primers. The cDNA was tested by PCR using specific

167

primers flanking an intron sequence to confirm the absence of genomic DNA

168

contamination and adjusted to threshold cycle (Ct) values within the mean range ±

169

1 of the reference gene to ensure similar cDNA yield for each qPCR reaction. All


using

a

modified

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of the cDNA was stored at -20°C.

171

Gene-specific PCR primers

172

Primer pairs used for quantitative PCR were designed using the software program

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PRIMER DESIGNER v. 2.0 (Scientific and Educational Software, Cary, NC) as

174

described previously,31 with melting temperatures (Tm) of 60 ± 2°C to facilitate

175

multi-parallel qPCR using a standard PCR program. Primer pairs flanking an intron

176

were compared by standard PCR using genomic DNA and cDNA as templates. The

177

specificity of each primer pair was analyzed by a melting curve at the end of the PCR

178

run, with one single peak revealing that the fluorescence signal was derived from the

179

intended amplicon with no dimer formation. PCR products were cloned into the

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pGEM T-easy vector (Promega, Madison, WI) and sequenced (Invitrogen, Shanghai,

181

China). Their size was checked on 1.5% agarose gels stained with ethidium bromide.

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The primer sequences, amplicon sizes, and Tm of all of the PCR products are

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indicated in Table 1.

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Quantitative real-time PCR (qRT-PCR)

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qRT-PCR was performed on three biological replicates per sample with the SYBR®

186

Green Supermix Kit (Bio-Rad, USA). The specific steps were as follows: each

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reaction was performed in a total volume of 12.5 µl, containing 0.625 µl (10

188

µmol/L) of each primer, 0.5 µl diluted cDNA, and 6.25 µl 2× SYBR® Green

189

Supermix and 4.5 µl nuclease-free water (TaKaRa, Japan) on a LightCycler 1.5



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(Roche, Germany). Initiation was by a preliminary step of 10 sec at 95°C, followed

191

by 45–50 cycles of 10 sec at 95°C for template denaturation, 20 sec at 60°C for

192

annealing, and 20 sec at 72°C for extension and fluorescence measurement.

193

No-template controls for each primer pair were included in each run. The

194

specificity of amplification was confirmed by melting curve analyses, and the

195

correct size of the amplification products was checked by the presence of a single

196

band of expected size for each primer pair in electrophoresis gels. Each reaction

197

was replicated three times.

198

Sequence Analysis and 3D Modeling

199

The software CLC Sequence Viewer v. 6.8 (CLC Bio, Aarhus, Denmark) was used

200

for multiple alignments of deduced amino acid sequences by using the built-in

201

standard progressive algorithm with the parameter set (gap open cost 10, gap

202

extension cost 0.1, and free end gap cost), and for molecular mass and isoelectric

203

point (pI) calculations. DNAStar MegAlign (Lasergene 6, Madison, WI) was used to

204

obtain the neighbor-joining phylogenetic tree and its bootstrap analysis by ClustalW

205

methods with the related parameter set as follows: gap penalty -10, gap length -0.1,

206

delay divergent seqs (%) -30, DNA transitions weight -0.5, protein weight

207

matrix-Gonnet 250, and bootstrap replicates -500. In silico predictions of signal

208

peptides

209

(www.cbs.dtu.dk/services/SignalP/) using both neural networks (NN) and hidden

210

Markov model (HMM) methods trained on eukaryotes and by setting the truncation

were

performed

with

the

SignalP


3.0

server

program32-33

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211

value to 70.

212

Three-dimensional (3D) structures were obtained by means of homology

213

modeling, and PDB ﬁles were retrieved with Phyre Web server v. 0.2.34 The 3D

214

models were drawn and rendered by means of UCSF Chimera for Microsoft Windows

215

64-bit35 (http://www.rbvi.ucsf.edu/chimera).

216

Known amino acid sequences used in the multiple alignment and phylogenetic

217

analysis, including Bet v 1 from European white birch (Betula verrucosa), Pru p 1 and

218

Pru p 2 from peach, Mal d 1 and Mal d 2 from apple (Malus domestica), Pru ar 1 from

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apricot (Prunus armeniaca), Pru av 1 and Pru av 2 from cherry (Prunus avium), Pyr c

220

1 from pear (Pyrus communis) and Jun a 3 from the mountain cedar (Juniperus ashei),

221

were retrieved from the National Center for Biotechnology Information (NCBI)

222

Database.

223

Statistical Analysis

224

The Ct values were determined for the target genes and the double internal control

225

genes Actin 2/7 (ACT, TC1223) and Translation elongation factor 2 (TEF2,

226

TC3544)36 with LightCycler® software 3.5 (Roche, Germany) using the second

227

derivative maximum method, and then they were exported to Microsoft Excel. The

228

relative expression of each gene was calculated using the comparative Ct method as

229

described by Thomas37 with the geometric average of the internal control genes used

230

as a normalization factor.38



231

RESULTS

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Bioinformatic Characterization of New Candidate Allergens

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Several cDNAs encoding yet unknown candidate allergens were found to have high

234

transcript rates in peach fruits. The most interesting sequences encode a new Bet v

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1-like isoform (Pru p 1.06B) and a thaumatin-like isoform (Pru p 2.01B), which have

236

been named according to the guidelines of the International Union of Immunological

237

Societies (IUIS, www.Allergen.org). Homology modeling of Pru p 1.06B and Pru p

238

2.01B deduced proteins that showed high structure similarity with known allergens

239

(Figures 1–2). The main calculated biochemical properties (molecular mass and pI)

240

and the presence of a signal peptide are reported in Table 2 for the both known and

241

candidate peach allergens (Supplementary Figure S2).

242

Multiple alignments of known Bet v 1-like amino acid sequences belonging to

243

Rosaceae species indicated a close similarity of Pru p 1.06B with Pru p 1.06A, Pru p

244

1.06C, and Pru av 1.02 (Figure 1A). Only one amino acid substitution (83, N/S) was

245

found between Pru p 1.06B and Pru p 1.06A. In addition, one substituted amino acid

246

(124, K/A) was found between Pru p 1.06B and Pru p 1.06C, while three substituted

247

amino acids (7S/A, 83N/S, and 124K/T) were found between Pru p 1.06B and Pru av

248

1.02. A neighbor-joining phylogenetic tree (Figure 3A) shows that Pru p 1.06B and

249

Pru av 1.02 were clustered in one group, and Pru p 1.06A and Pru p 1.06C are

250

grouped together. Both groups are somewhat distant from Pru p 1.01, Pru p 1.02, Pru

251

p 1.03, and Pru p 1.04.


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According to multiple alignments of known thaumatin-like proteins (TLPs), the

253

new candidate allergenic protein Pru p 2.01B showed only 7 unique amino acids out

254

of 246 with known Pru p 2.01A (Figure 2A). In the phylogenetic analysis, Pru p

255

2.01B and Pru p 2.01A were clustered in one group, which was very close to apple

256

Mal d 2.01A and Mal d 2.01B, whereas another peach TLP member, Pru p 2.02, was

257

more similar to the Pru av 2 allergen of cherries. Moreover, with regard to the 3D

258

structures, a close similarity with cherry Pru av 2 was observed (Figure 2B, C),

259

indicative of comparable allergenic properties.

260

Effect of fruit bagging

261

Gene expression profiles of the major peach allergen-encoding genes were determined

262

for both bagged fruits and the control fruits. The results are summarized in Figure

263

4A–D. Fruit bagging had a significant effect on the expression of selected Pru p 1

264

genes as shown in Figure 4A. For Pru p 1.01, a decreasing trend in transcript

265

accumulation was observed in epicarps of bagged fruit, whereas the opposite effect

266

was observed in the mesocarp. Concerning Pru p 1.06B, a clear down-regulation by

267

bagging treatment was also observed in the epicarp, whereas no significant difference

268

was observed in the mesocarp.

269

As far as Pru p 2 genes are concerned, fruit bagging significantly decreased the

270

transcriptional rate of Pru p 2.01B in the epicarp, while no significant difference was

271

observed in the mesocarp. For Pru p 2.02, the expression levels were downregulated

272

by bagging treatment in both the epicarp and mesocarp (Figure 4B).



273

In the case of the Pru p 3.01 gene (Figure 4C), transcription in the epicarp was

274

much higher than in the mesocarp by an average of 3 log. Bagging with opaque paper

275

significantly downregulated Pru p 3.01 expression in both the mesocarp and epicarp.

276

Pru p 3.01 was expressed in the mesocarp at a very low and almost undetectable level,

277

especially in the mesocarp of bagged fruits.

278

For the two profilin-encoding genes, the same trend was found. Bagging

279

significantly reduced Pru p 4.01 and Pru p 4.02 transcription in the epicarp, whereas

280

the opposite effect was observed in the mesocarp (Figure 4D). Interestingly, in the

281

control fruits, the expression of the two genes in the epicarp was much higher than in

282

the mesocarp, but for the bagged fruits, a 2-fold higher expression in the mesocarp

283

was observed compared to the epicarp.

284

Considering the total protein content of peach fruit, a significant effect of bagging

285

in a tissue-dependent manner was pointed out (Figure 5B). The total protein content

286

of the peach epicarp was significantly reduced by bagging treatment, whereas the

287

opposite effect was observed in the mesocarp.

288

In addition to the transcript level of allergen-encoding genes, the influence of

289

fruit bagging with opaque paper on peach maturity-related quality was also analyzed.

290

The skin color was significantly affected by the bagging treatment (Figure 5A). The

291

non-bagged fruits were found to have a full and dark red color, whereas the fruits

292

covered with opaque paper revealed a light yellow color with very little pigment

293

accumulation. The weight of the peaches covered with opaque paper bags was

294

significantly higher than the non-bagged fruit. Values of fruit firmness were slightly


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higher in the control peaches than the bagged ones. A significant decrease of the TSS

296

content was observed in the bagged fruit, whereas the TA content in the bagged

297

peaches was slightly lower (Table 3).

298

DISCUSSION

299

In the present study, two newly identified candidate allergens, Pru p 1.06B and Pru p

300

2.01B, were characterized for the first time. In a previous study, Yang et al.11 found

301

that Pru p 1.06B is one of the two most abundant candidate allergens (Pru p 1.01 and

302

Pru p 1.06B) among the Pru p 1 family in peaches, while Pru p 2.01B is the

303

predominant member of the Pru p 2 family, indicating that these two candidate

304

allergens may play key roles in allergic reactions to peaches. The multiple sequence

305

alignment results obtained by bioinformatics analysis indicate a close structural

306

similarity between these two candidate allergens and the corresponding proteins found

307

in other Rosaceae species, such as apple, cherry, pear, and apricot. Therefore, we can

308

assume that we have identified a new Bet v 1-like (Pru p 1.06B) and a TLP-like (Pru p

309

2.01B) allergen in peach.

310

Fruit bagging is currently widely used on fruit trees to produce unblemished,

311

high-quality fruits. The practice not only decreases the quantity of light but it also

312

alters the quality of light to various extents and may affect other environmental

313

factors.39-40 In our study, fruit bagging was used as a method of fruit shade stress to

314

explore how shading during fruit growth and development affect the gene expression

315

of major peach allergens. The experimental results showed that bagging with opaque



316

paper during fruit growth and development had a significant effect on major peach

317

allergen-encoding genes, as shown in Figure 4A–D.

318

Among Pru p 1 genes, low light had a significant effect on Pru p 1.01 and Pru p

319

1.06B. A clear and significant interaction was observed between bagging and tissue

320

expression for both genes. Light deprivation downregulated the expression of Pru p

321

1.01 at the transcript level in the epicarp, but usually to a lesser extent than in the

322

mesocarp and with an opposite trend (Figure 4A). These results are consistent with

323

the findings of Botton, who observed that in apples, shadowing affected the gene

324

expression of Mal d 1.01 and 1.02 in a tissue-dependent manner, with the genes being

325

down- and up-regulated in the cortex and epidermis, respectively.14 The same group

326

also found that light enhancement up-regulated Pru p 1.01 in the epicarp but

327

decreased it in the pulp.18 In the present research, the expression pattern of Pru p

328

1.06B under different light conditions was studied for the first time, and the same

329

trend with Pru p 1.01 was found for Pru p 1.06B, with the only exception that Pru p

330

1.06B remained almost constant in the mesocarp of the bagged and control fruits

331

(Figure 4A).

332

In the Pru p 2 class, a significant effect of bagging on the transcription of Pru p

333

2.01B and 2.02 genes was discovered. A decreasing trend in the transcript

334

accumulation of Pru p 2.02 was observed in both the mesocarp and epicarp, whereas

335

for Pru p 2.01B, the gene was up-regulated in the epicarp and no significant

336

difference was detected in the mesocarp (Figure 4B). In previous studies, Botton

337

found that in apples light deprivation had no significant effect on the transcription of


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338

the Mal d 2 class, except for Mal d 2.02, which was affected in a tissue-dependent

339

manner, and the transcription was inhibited in the cortex and stimulated in the

340

epidermis.14 Light enhancement has been found to have no effect on Pru p 2.01

341

transcript accumulation.18

342

In the case of the Pru p 3.01 gene, a clear negative relation was shown in both

343

the mesocarp and epicarp between fruit bagging and transcript accumulation (Figure

344

4C). Fruit bagging significantly reduced Pru p 3.01 transcription, regardless of the

345

tissue. This is consistent with Botton’s findings, which indicated that light

346

enhancement can significantly upregulate Pru p 3.01 gene transcription, especially in

347

lower fruit load samples18. Interestingly, Botton et al. also demonstrated that in apples,

348

shadowing significantly increased Mal d 3.01 and Mal d 3.02 transcription in the

349

epidermis, whereas there was an opposite effect in the cortex.14

350

In the two profilin-encoding genes, the effect of fruit bagging was significant,

351

and both Pru p 4.01 and 4.02 were dependent on tissue. Clear upregulation was

352

observed in the mesocarp, while there was an opposite effect in the epicarp (Figure

353

4D). In a previous study, it was suggested that shadowing with plastic netting

354

(decreasing the light radiation by ∼30%) had no significant effect on apple

355

profilin-encoding gene Mal d 4.01 transcription, but the treatment enhanced the gene

356

expression of Mal d 4.02 in the skin.14 Botton et al. found that light enhancement only

357

slightly reduced Pru p 4.02 transcription in both the mesocarp and epicarp, and it had

358

no significant effect on the transcriptional accumulation of Pru p 4.01.18

359

This study is the first to use gene expression profiling to reveal the effect of



360

bagging with light-impermeable paper bags on peach allergens. Light deprivation was

361

shown to significantly affect the expression of the allergen-related genes. A

362

significant reduction of the total protein content was also observed in the epicarp of

363

the bagged fruit (Figure 5B), which might suggest a significant decrease of peach

364

allergens at the protein level in the bagged epicarp to a certain extent. Several studies

365

indicated that fruit photosynthesis can contribute a part of their total growth

366

carbohydrate requirements, and green peels showed remarkable photosynthetic

367

activity, as reviewed by Blanke et al.41 Lytovchenko et al. suggested that one of the

368

functions of tomato fruit photosynthesis is providing an important source of carbon

369

assimilate for properly timed seed development.42 Chen et al. demonstrated that the

370

peel photosynthesis in Satsuma mandarin fruit plays an important role in the

371

development of the peel itself.43 Chen et al. also reported that the sun-exposed peel of

372

apple fruit has a higher photosynthetic capacity than the shaded peel.44 However,

373

there is very little information in the literature concerning photosynthesis of peach

374

fruit. In a previous study, Pavel and DeJong estimated that the total photosynthetic

375

contribution of peach fruits to their carbohydrate budget amounts to 5–9% depending

376

on the fruit’s exposure to light.45 However, it is still not clear how the peel

377

photosynthates of peach fruits are translocated and consumed. Fruit bagged with

378

opaque paper growing in an almost-dark condition and the photosynthetic activity of

379

the peach fruit itself might be influenced. Moreover, some studies have found that the

380

allergenic composition of peach fruit is mainly located in the peel.11, 46-47 Taking into

381

account all of these aspects, we speculated that the significant reduction in the


Page 18 of 46

Page 19 of 46


382

expression of allergen-encoding genes in bagged peach peels is related to the

383

suppression of photosynthesis in fruit peels. We hypothesized that the bagging

384

treatment reduced the light irradiation of the bagged peach fruit, decreased the

385

photosynthetic activity of the peach peel, and affected the allergen-encoding gene

386

expression pattern. However, since the synthesis and degradation mechanism of these

387

peach allergens remains unclear at present, further study is needed to clarify the

388

specific mechanism responsible for these effects.

389

In addition, previous studies have demonstrated that a considerable percentage of

390

identified plant allergens can be group into the pathogenesis-related protein

391

(PR-protein) families.48 Zubini et al. found that the two isforms of Pru p 1, Pru p

392

1.01 and Pru p 1.06D, play an important role in peach defense to the fungal pathogen

393

Monilinia spp.49 Pru p 2 was reported to be involved in the resistance of peach fruit

394

to bacterial spot disease caused by Xanthomonas campestris pv. pruni (Xcp).50 These

395

PR-proteins are constitutively expressed in some organs and they can be induced to a

396

significantly higher degree by pathogen attack. As a physical protection technique,

397

fruit bagging has been reported to protect fruits from various pathogens and

398

mechanical damage.51-52 In this study, non-bagged peach fruits were more

399

susceptible to infection by pathogens. Therefore, we assume that the significant

400

reduction in the expression level of allergen-encoding genes in bagged peach fruits,

401

especially for the isforms of Pru p 1 and Pru p 2, might be related to the protective

402

effect of fruit bags. Different gene-silencing strategies have been applied to decrease

403

the accumulation of allergenic proteins in soybean,53 rice,54 apple,55 and tomato56 to



404

create low-allergenic food sources. Although there is no related report yet on these

405

trangenic plants about the strength of defense against microbial pathogens and other

406

stress-causing stimuli, reduced allergen accumulation theoretically might have some

407

certain unwanted impact on their resistance, which still needs further study. To

408

address this problem, Le et al.56 proposed that genetic manipulation should be

409

restricted to target tissues by selecting the appropriate fruit-, seed-, or pollen-specific

410

promoter sequences. Le et al. also suggested that silencing approaches should be

411

combined with the expression of hypoallergenic variants of the same protein to

412

restore physiologic function. Compared to these silencing approaches, bagging is a

413

relatively simple fruit-specific method that can be potentially used to reduce the

414

allergenic component in peach fruit.

415

It is noteworthy that fruit bagging not only affected the transcriptional expression

416

of allergen-encoding genes in peach fruit, but also had a certain effect on fruit

417

quality, especially on fruit skin coloration and TSS content (Figure 5A and Table 3),

418

which significantly influence the acceptability of the peach fruit. In this study, fruit

419

bagged with opaque paper were distinctly paler than the non-bagged ones. Previous

420

studies have demonstrated that the accumulation of anthocyanin in fruit skin is

421

positively related to the light transmittance of the paper bags.21-23 It has been pointed

422

out that anthocyanin concentrations in the peel of fruit increased rapidly after bag

423

removal prior to harvest.22-23,

424

significantly lower TSS content, which is in line with a previous study by Zhang et

425

al.57 Based on the data, to obtain peach fruit with an attractive appearance and good

57

Furthermore, the bagged peach fruits had a


Page 20 of 46

Page 21 of 46


426

inner qualities, fruit should be covered with light-impermeable bags at the early

427

stage of fruit development and the bag should be completely removed at least 7 days

428

before harvest.

429

In summary, fruit bagging with opaque paper significantly reduced the

430

expression of peach allergen-encoding genes, and it can be potentially used as an

431

effective agricultural practice to reduce peach allergen content in a fruit-specific

432

manner. However, bagging until harvest may bring some adverse effects on the

433

appearance and taste of the fruit, so further improvement of the bagging treatment,

434

such as bag removal in advance, should be considered.

435

ACKNOWLEDGMENTS

436

This research was supported by the Natural Science Foundation of China (31700604),

437

Key Scientific Research Project of Henan Higher Education (16A210011), and Key

438

Technology Project of Henan Province (172102110106 and 162102110160). We

439

thank Hongwei Ren from Luoyang Academy of Agriculture and Forestry for

440

providing the local peach materials and assistance in sample treatment.

441

ABBREVIATIONS USED

442

nsLTP,

443

thaumatin-like protein; 1-MCP, 1-methylcyclopropene; GAP, good agricultural

444

practice; pI, isoelectric point; AMV, alfalfa mosaic virus; qRT-PCR, quantitative

445

real-time PCR; TSS, total soluble solids; TA, titratable acidity; Ct, threshold cycle;

nonspecific

lipid

transfer

protein;

PR,


pathogenesis-related;

TLP,


446

LD, longitudinal diameter; HD, horizontal diameter; ACT, Actin 2/7; TEF2,

447

Translation elongation factor 2

448

SUPPORTING INFORMATION

449

The supplementary information are available in the Web edition of the Journal.

450

Supplementary Figure S1. Correlation analysis results of the expression patterns of

451

peach major allergen-encoding genes in the two seasons (2016 and 2017).

452

Supplementary Figure S2. Integral multiple alignments of Rosaceae TLP amino acid

453

sequences including the predicted signal peptide and the dominant IgE epitope.

454 455 456 457 458 459 460 461 462 463 464 465 466


Page 22 of 46

Page 23 of 46


467

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56. Le, L.Q., et al., Reduced allergenicity of tomato fruits harvested from Lyc e

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57. Zhang, B.B., et al., Relationship between the bagging microenvironment and fruit

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58. Leone, P., et al., Resolution of the structure of the allergenic and antifungal

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Page 30 of 46

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629

Figure Legends

630



631

Figure 1. (A) Multiple alignments of birch Bet v 1 and Rosaceae Bet v 1-like

632

amino acid sequences. The newly identified peach Bet v 1-like allergen (Pru p

633

1.06B) is included. The bar chart below the alignment indicates the amino acid

634

conservation. Nonconserved amino acids are marked in different colors, and the

635

consensus sequence displays 100% conserved residues. The black arrows above

636

the alignment and the box indicate the IgE epitope. Accession numbers are

637

reported where available. (B) Three-dimensional model of Pru p 1.06B allergen.

638

(C) Three-dimensional model of known Pru p 1.02 allergen. The putative

639

epitopes are marked in different colors. The corresponding amino acid residues

640

are also indicated.

641


Page 32 of 46

Page 33 of 46


642

Figure 2. (A) Multiple alignments of Rosaceae TLP amino acid sequences,

643

including TLPs from peach, as well as Pru av 2 from cherry (Prunus avium), Jun

644

a 3 from the mountain cedar (Juniperus ashei), and Mal d 2.01A and Mal d 2.01B

645

from apple (Malus domestica). Only the putative epitope region is shown. The bar

646

chart below the alignment indicates the amino acid conservation. Nonconserved

647

amino acids are shaded, and the consensus sequence displays the 100%

648

conserved residues. The dominant IgE epitope is noted below the sequence

649

according to Leone et al.58 (B) Three-dimensional model of Pru p 2.01B. (C)

650

Three-dimensional model of known cherry TLP Pru av 2. The epitope pointed

651

out in (A) is marked in red.

652



653

Figure 3. Neighbor-joining phylogenetic trees of the sequences aligned in Figures

654

1A (A) and 2A (B). Bet v 1 and Jun a 3 were used as the outgroup in the two NJ

655

trees, respectively. Amino acid substitutions are reported below the trees and

656

accession numbers are displayed in parentheses.


Page 34 of 46

Page 35 of 46


657



658

Figure 4. Expression profiles of Pru p 1.01, Pru p 1.06B (A), Pru p 2.01B, Pru p

659

2.02 (B), Pru p 3.01 (C), Pru p 4.01, and Pru p 4.02. (D) Genes in mesocarp and

660

epicarp of fruits grown in standard conditions (non-bagged) and fruits bagged

661

with opaque paper. GM (TEF2 *ACT) means the geometric average of the

662

expression of the two internal control genes TEF2 and ACT. The letters represent

663

the nonsignificant ranges according to the LSD test (P ≤ 0.05). Bars represent the

664

standard error.


Page 36 of 46

Page 37 of 46


665 666

Figure 5. (A) Non-bagged ‘Zaochunhong’ peach fruit as the control (a) and

667

‘Zaochunhong’ peach fruit bagged with opaque paper (b). (B) The total soluble

668

protein contents of peach fruits: NE - the epicarp of non-bagged peaches; BE -

669

the epicarp of bagged peaches; NM – the mesocarp of non-bagged peaches; BM –

670

the mesocarp of bagged peaches. The letters represent the nonsignificant ranges

671

according to the LSD test (P ≤ 0.05). Bars represent the standard error.



Page 38 of 46

Table 1. Gene-specific primers used for real-time RT-PCR Amplicon Size (bp)

Flanking regionb

TCCTTCAAGGATTTCAGAATG

121

1

AAAGCCCTTGTTCTTGAAGCG

CATCTCCTTCAACCAAAGTGTAGT

211

‘1/2

EU424259

AACAAAGTGTGCCCGGCTCC

TCTCCGGCTTGTCGTTAGGT

127

2

Pru p 2.02

EU424255

CATGTGCAACGGTAAGACTG

CGCTCCCATCGGACCCTATC

227

2

Pru p 3.01

EU424265

CTTTGGTGGTGGCCTTGT

CTCACGTAGGGTATGCATGG

103

1

Pru p 4.01

EU424271

AGCAGTACGTCGATGACCAC

ACCGGCTTCACCTTGGATCA

230

‘1/2

Pru p 4.02

EU424273

GTAGACGACCATCTGATGTG

TGTGCCTCCAAGATACAACC

192

‘1/2

ACT

TC1223

GTTATTCTTCATCGGCGTCTTCG

CTTCACCATTCCAGTTCCATTGTC

112

-

TEF2

TC3544a

GGTGTGACGATGAAGAGTGATG

TGAAGGAGAGGGAAGGTGAAAG

129

-

Gene

Acc no.

Forward Primer [5’-3’]

Reverse Primer [5’-3’]

Pru p 1.01

EU424240

GAGCGAGTTCACCTCTGAGA

Pru p 1.06B

EU424250

Pru p 2.01B

a

b

a

Peach EST database accession number. Numbers refer to the position of exons to which the primers belong.


Page 39 of 46


Table 2 Classification of the Main Peach Allergensa Allergen class

Allergen/Isoallergen

Synonym

Nucleotide acc. no.

Mol mass (kDa)

pl

Signal peptide

Pru p 1

Pru p 1.01

ypr-10

EU424240/DQ251187

17.648

6.02

No

Pru p 1.02

AM290651

17.4

5.26

No

cPru p 1.06B

EU424250

17.437

5.36

No

AF362988

25.8

8.64

No

EU424259

25.81

8.76

Yes

Pru p 2

Pru p 2.01

PpAz44

cPru p 2.01B

a

Pru p 2.02

PpAz8

EU424255

25.65

5.1

Yes

Pru p 3

Pru p 3.01

PpLTP1

EU424265

11.8

9.51

Yes

Pru p 4

Pru p 4.01

EU424271

14.061

4.97

No

Pru p 4.02

EU424273

14.019

4.98

No

All of the parameters were calculated for the mature proteins. Accession numbers are given as a reference and do not always refer to full-length

sequences (“c” before the name means “candidate”).



Page 40 of 46

Table 3 Effects of bagging treatment with opaque paper on the fresh weight (Weight), longitudinal diameter (LD), horizontal diameter (HD), firmness, total soluble solids (TSS), and titratable acidity (TA) of peach fruits at harvesta,b

a

Non-bagged Fruit

Bagged Fruit

Weight (g)

145.63 ± 4.88b

156.42 ± 6.79a

LD (cm)

7.05 ± 0.18a

6.92 ± 0.28a

HD (cm)

6.42 ± 0.22a

6.67 ± 0.13a

Firmness (N)

42.8 ± 1.55a

40.25 ± 1.65a

TSS (°Brix)

11.75 ± 0.12a

10.65 ± 0.17b

TA (% malic acid)

0.193 ± 0.0036a

0.178 ± 0.0045a

Each value represents the mean ± standard error of three replicates. b

Values indicated by different letters are significantly different to controls (LSD, 0.05).


Page 41 of 46 A



A.


IgE epitope


Page 42 of 46

Page 43 of 46


A

B


Amino acid substitutions (x100)

Bagged Journal ofNon-bagged Agricultural and Food Chemistry 1.0E+03

Pru p 1.01 Mean Normalized Expression (A.U.) Ratio target gene / GM (TEF2 * ACT)

1.0E+03 Mean Normalized Expression (A.U.) Ratio target gene / GM (TEF2 * ACT)

A

a 1.0E+02 b c 1.0E+01 d 1.0E+00

1.0E-01

1.0E+02 b

1.0E+01

c

c

1.0E+00 1.0E-01 1.0E-02

Mesocarp

1.0E+02 1.0E+01 a

a

1.0E+00 1.0E-01

b

1.0E-02

1.0E+03 Mean Normalized Expression (A.U.) Ratio target gene / GM (TEF2 * ACT)

a

c c

c

Mesocarp

Pru p 4.02

1.0E+02 a

b

1.0E+01 c d 1.0E+00

Epicarp

Epicarp

Mesocarp

1.0E+03

Pru p 3.01 a

Mean Normalized Expression (A.U.) Ratio target gene / GM (TEF2 * ACT)

Mean Normalized Expression (A.U.) Ratio target gene / GM (TEF2 * ACT)

c

Pru p 2.02

Epicarp

Pru p 4.01

1.0E+00

C

c

1.0E+00

Mesocarp

b 1.0E+01

1.0E+01

1.0E-03 Epicarp

1.0E+02

b

1.0E+03

Pru p 2.01B a

1.0E+03

1.0E+02

Epicarp

1.0E-03

D

a

Mesocarp

Mean Normalized Expression(A.U.) Ratio target gene/GM(TEF2*ACT)

Mean Normalized Expression(A.U.) Ratio target gene/GM(TEF2*ACT)

1.0E+03

Pru p 1.06B

1.0E-01 Epicarp

B

Page 44 of 46

1.0E+02 b 1.0E+01 c

1.0E+00 1.0E-01

d 1.0E-02

ACS Paragon Plus Environment Epicarp

Mesocarp

Mesocarp

Page 45 of 46


A

(a)

B

1.4E+03

(b) a

Total soluble protein content (Pg¦g-1 FW)

1.2E+03

b

1.0E+03 C 8.0E+02 d 6.0E+02

4.0E+02 2.0E+02 0.0E+00 NE

BE


NM

BM


Graphic For Table of Contents Only 238x173mm (96 x 96 DPI)


Page 46 of 46

Significant Reduction of the Expression of Peach (Prunus persica L

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