Distinct Carotenoid and Flavonoid Accumulation in a Spontaneous

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Distinct carotenoid and flavonoid accumulation in a spontaneous mutant of Ponkan (Citrus reticulata Blanco) results in yellowish fruit and enhanced postharvest resistance Tao Luo, Kunyang Xu, Yi Luo, Jiajing Chen, Ling Sheng, Jinqiu Wang, Jingwen Han, Yunliu Zeng, Juan Xu, Jianmin Chen, Qun Wu, Yunjiang Cheng, and Xiuxin Deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02807 • Publication Date (Web): 02 Sep 2015 Downloaded from http://pubs.acs.org on September 5, 2015

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Journal of Agricultural and Food Chemistry

Wu, Qun; Quzhou Bureau of Agriculture Economic Specialty Station, Cheng, Yunjiang; Key Laboratory of Horticultural Plant Biology (Ministry of Education) and Key Laboratory of Horticultural Crop Biology and Genetic Improvement, Central Region (Ministry of Agriculture), Huazhong Agricultural University Deng, Xiuxin; Key Laboratory of Horticultural Plant Biology (Ministry of Education) and Key Laboratory of Horticultural Crop Biology and Genetic Improvement, Central Region (Ministry of Agriculture), Huazhong Agricultural University

ACS Paragon Plus Environment


1

Distinct carotenoid and flavonoid accumulation in a spontaneous mutant of Ponkan

2

(Citrus reticulata Blanco) results in yellowish fruit and enhanced postharvest

3

resistance

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Tao Luo,† Kunyang Xu,† Yi Luo,† Jiajing Chen,† Ling Sheng,† Jinqiu Wang,† Jingwen

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Han,† Yunliu Zeng,† Juan Xu,† Jianmin Chen,‡ Qun Wu,‡ Yunjiang Cheng,*,† Xiuxin

6

Deng†

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†

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Laboratory of Horticultural Crop Biology and Genetic Improvement, Central Region

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(Ministry of Agriculture), Huazhong Agricultural University, Wuhan 430070, People’s

Key Laboratory of Horticultural Plant Biology (Ministry of Education) and Key

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Republic of China

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‡

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Province, People’s Republic of China

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*Corresponding author (Phone: +86-2787281796; Fax: +86-2787280622; Email:

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[email protected])

Quzhou Bureau of Agriculture Economic Specialty Station, Quzhou 324000, Zhejiang

1 ACS Paragon Plus Environment

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ABSTRACT

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As the most important fresh fruit worldwide, citrus is often subjected to huge postharvest

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losses caused by abiotic and biotic stresses. As a promising strategy to reduce postharvest

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losses, enhancing natural defense by potential metabolism reprogramming in citrus

19

mutants has rarely been reported. The yellowish spontaneous mutant of Ponkan (Citrus

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reticulata Blanco) (YP) was used to investigate the influence of metabolism

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reprogramming on postharvest performance. Our results show that the reduced xanthophyll

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accumulation is the cause of yellowish coloring of YP and might be attributed to the

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reduced carotenoid sequestration capacity and up-regulated expression of carotenoid

24

cleavage dioxygenase genes. Constantly higher levels of polymethoxylated flavones

25

(PMFs) during the infection and the storage stage might make significant contribution to

26

the more strongly induced resistance against Penicillium digitatum and lower rotting rate.

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The present study demonstrates the feasibility of applying bud mutants to improve the

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postharvest performance of citrus fruits.

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Key words: C. reticulata Blanco, yellowish mutant, carotenoids, flavonoid, Penicillium

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digitatum, postharvest resistance



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INTRODUCTION

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Carotenoids are a class of C40 lipophilic isoprenoids highly conserved throughout life

33

evolution. As the second abundant naturally occurring pigments, carotenoids are

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synthesized not only in photosynthetic organisms (bacteria, algae and plants) but also in

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non-photosynthetic bacteria and fungi.1 Due to their diversity, carotenoids impart various

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colors to these organisms. In addition, carotenoids are indispensable secondary metabolites

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involved in photosynthesis, antioxidation, and phytohormone biosynthesis in plants.2 Plant

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carotenoid biosynthesis has been well elucidated and is initiated with the yield of phytoene

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by condensation of two geranylgeranyl diphosphate molecules, which are derived from C5

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isopentenyl diphosphate (IPP) in plastidial methylerythritol 4-phosphate (MEP) pathway.3

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Subsequently, the red lycopene is generated from phytoene through two-step desaturation

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and isomerization. After that, diversified forms of carotenoids are generated by cyclization,

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hydroxylation, epoxidation, cleavage and other modifications.1 (Fig. S1).

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The carotenoids in colorful organs or organisms are the results of steady-state

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carotenoid accumulation, which is dependent on the metabolic equilibrium between

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biosynthesis and degradation along with storage.1,4 The crucial rate-controlling factor

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phytoene synthase (PSY) influences the carotenoid content by varying its expression or

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activity.5-7 The critical cyclases, lycopene β-cyclase (LCYB) and lycopene ε-cyclase

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(LCYE), are important in determining carotenoid content and the β-carotene/α-carotene

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ratio.8 Carotenoid content was shown to be positively correlated with the up-regulation of

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β-carotene hydroxylase (BCH) gene induced by sugar in potato tubers.9 BCH was also

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proved to determine the levels of both alpha-carotene and total carotenoid in orange

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carrots.10 Carotenoid accumulation would be enhanced by the increase of plastid

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compartment size (hp1/2/3; Golden 2-like)11-14 and the sequestration capacity of


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carotenoids (CHRC; fibrillin protein)15-17 and by the posttranslational modification on PSY

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protein.18 Xanthophylls are a class of mono- or dihydroxylated carotenoids which are

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usually esterified with various fatty acids during the ripening of the fruits.19-20 Xanthophyll

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esterification was proved to be a constitutive process accompanying carotenoid

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overaccumulation in chromoplast,20 while the carotenoid modifying gene PYP1 (PALE

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YELLOW PETAL 1), which plays an essential role in xanthophyll esterification, was

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recently reported to influence carotenoid content and chromoplastid development.21

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Carotenoids are usually cleaved by carotenoid cleavage dioxygenases (CCDs), thus the

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expression of CCDs (e.g. CCD1&CCD4) was found to be negatively correlated with

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carotenoid accumulation in various plant species or tissues.3,

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CCD1 expression does not alter violaxanthin accumulation in citrus. 1, 26 It is worthy to

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note that CCD4 in citrus (CitCCD4) was proved to produce a fruit-specific C30

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apocarotenoid (β-citraurin).27-28

22-25

However, increased

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As the most important fresh fruits, citrus fruits are a vital source of dietary

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carotenoids.29 Spontaneous bud mutation occurs occasionally in citrus and facilitates the

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development of new varieties.30 A considerable number of citrus bud mutants have been

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studied to investigate the mechanism of carotenoid accumulation. For example, Pinalate, a

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yellowish mutant of Navelate orange (Citrus sinensis L. Osbeck), accumulates linear

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carotenes; 31 and both Hong Anliu, a red-flesh mutant of sweet orange (Citrus sinensis L.

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Osbeck),30 and Cara Cara, a red-flesh mutant of Navel orange (Citrus sinensis L.

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Osbeck),32 accumulate lycopene. However, very few studies have been focused on other

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biological processes which may be affected by the altered carotenoid profile in these

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mutants.

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Similar to other perishable agricultural products, citrus fruits are susceptible to the



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damage caused by physical disorder and pathogenic diseases during shipping, storing and

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marketing.33 Citrus green mould rot, which is caused by Penicillium digitatum, leads to

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huge postharvest losses in citrus industry.34 Although the application of artificial fungicide

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was proved to be inexpensive and effective to control this disease, it is greatly limited by

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the emergence of new resistant fungal strains as well as by its low potency, poisonousness,

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poor solubility and nonbiodegradability.34-35 Enhancing the natural defense of the fruit by

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reprogramming its physiological status would be a promising alternative strategy in the

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prevention of this pathogen.33-35 Flavonoids are a group of polyphenolic compounds that

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include flavanone, flavanone- and flavone-O-glycosides, flavone-C-glycosides, flavone

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and polymethoxy flavone aglycones.35 The PMFs in citrus are a class of flavones found in

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glycosylated and aglycone states, showing a great variety of compounds with their

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structure frequently multisubstituted by hydroxyl and/or methoxyl groups.36 Flavonoids

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especially polymethoxylated flavones (PMFs), which are rich in citrus peel, have been

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proved to be essential barriers against pathogen attack and can be induced to accumulate in

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citrus fruits.34, 36-38

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A non-targeted metabolomic analysis revealed significantly different primary and

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secondary metabolites in fruits between the bud mutant ‘Hong Anliu’ and its parent.39

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Enhanced carotenoid accumulation in citrus calli by over-expressing CrtB affected redox

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status and starch metabolism, and was linked with flavonoid/anthocyanin accumulation.2

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These results indicate that the altered carotenoid accumulation might affect other

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metabolisms potentially associated with carotenoids. However, no studies have tested the

100

influences of the carotenoid-associated changes of metabolism on the abiotic and biotic

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stress resistance in fruits of these citrus mutants. A yellowish bud mutant has been

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identified from Ponkan (C. reticulata Blanco), which has reduced xanthophyll


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accumulation in fruits. Here, we used this bud mutant to explore the possible alterations in

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primary and secondary metabolisms, especially flavonoid accumulation, which is strongly

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related to the resistance against abiotic and biotic stresses. The feasibility of applying this

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bud mutant to improve postharvest performance of fruits was further tested by storage

107

experiment and inoculation with P. digitatum.

108 109

MATERIALS AND METHODS

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Plant materials. The yellowish bud mutant of Ponkan (C. reticulata Blanco) (PK) was

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found in year 2008 by Quzhou Bureau of Agriculture Economic Specialty Station from the

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grafted seedlings of PK in orchard. The yellowish coloring in fruits of YP was confirmed

113

to be stable by an investigation of six years. The materials of YP and PK used for the

114

present study were grown following commercial cultivation practices in the same orchard

115

in Quzhou, Zhejiang Province, China. Ripening fruits were harvested on 10th (coloring

116

stage 1, CS1), 20th (coloring stage 2, CS2) and 30th (coloring stage 3, CS3) November in

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the year of 2013 and 2014.

118

Thirty fruits as one sample were peeled and immediately frozen in liquid nitrogen. One

119

half of the pulp was immediately frozen in liquid nitrogen while the other half was used for

120

quality analysis. All samplings were performed in three biological repeats. Frozen samples

121

were kept at -80°C until use. The transverse and longitudinal diameters of leaves were

122

determined by a vernier caliper.

123

Chemicals. Acetonitrile, methanol, formic acid and authentic standards of flavonoids were

124

of HPLC grade and were provided as follows: acetonitrile and methanol by Fisher (Fisher

125

Scientific, USA), formic acid by Guangfu (Tianjin Guangfu Fine Chemical Research

126

Institute, China), Eriocitrin (ERI), Narirutin (NART), Hesperidin (HES), Didymin (DID),



127

Nobiletin (NOB) and Tangeretin (TAN) (purity ≥ 98%) by YiFang S&T (Tianjin, China),

128

Sinensetin (SIN) from Chromadex (Santa Ana, CA, USA), and 5-demethyl-nobiletin

129

(DEM) from MeiLian (Shanghai, China). Deionized water was prepared by distilled water

130

through a Milli-Q A10 system (Millipore, Milford, MA, USA).

131

Color measurement. Fruit color was measured by a color analyzer (KONICA

132

MINOLTA CR-400, Japan). The red to green was expressed as +a to –a, yellow to blue

133

was expressed as +b to –b, and brightness was expressed as L. The color index (CI) was

134

calculated by the equation (1): CI =

135

(1)

136

Analysis of titratable acid and total soluble solids. Hand squeezed juice from sliced pulp

137

was filtrated and subjected to titratable acid (TA) and (total soluble solids) TSS analysis.

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TA was determined by a Fruit Acidity Meter (GMK-835F, G-WON HITECH CO., LTD,

139

Korea) and TSS by a Brix Refractometer (Pocket PAL-1, ATAGO, Japan).

140

Carotenoid extraction and HPLC analysis. Carotenoid extraction and analysis by

141

reversed-phase high-performance liquid chromatography (RP-HPLC) were performed

142

according to a previous work.31 Retention time and UV spectrum of isolated carotenoids

143

were listed in Supplementary Table 1.

144

Analysis of abscisic acid. Abscisic acid (ABA) was extracted from 1.0 g fresh sample

145

with 4 mL extraction solution (MeOH: water = 4:1, contain 1mM BHT, 2,

146

6-Di-tert-butyl-4-methyl- phenol) at 4 °C as previously described.40 Samples and standard

147

solutions were analyzed by the indirect enzyme-linked immunosorbent assay (ELISA). The

148

ELISA kit was kindly supplied by Professor Baomin Wang (China Agricultural

149

University).

150

Analysis of primary metabolites by GC-MS. Primary metabolites and secondary


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metabolites were extracted and analyzed by GC-MS as previously described.33

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Flavonoid analysis by UPLC. 1.0 g flavedo or 4.0 g pulp was grinded by liquid nitrogen

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and extracted using 80% MeOH for more than three times. After centrifugation, the

154

supernatants were collected and metered volume to 25 mL for flavedo and to 10 mL for

155

pulp. After the filtering through a 0.22 um filtration membrane (Millipore, USA), 2 ul

156

sample was injected and analyzed in a Waters H-Class UPLC system (eλ PDA detector,

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HSS T3 column: 150 mm×3.0 mm i.d., 1.8 um particle, Waters, USA). Optimum mobile

158

phase was composed of phase A (water: acetonitrile: formic acid = 100: 2: 0.1) and phase

159

B (acetonitrile containing 0.1% formic acid). The elution program was performed as

160

follows (min, % A): (0, 100), (2, 95), (8, 75), (12, 55), (20, 25), (23, 0). The equilibration

161

time after gradient analysis was 5 min. The flow rate was 0.5 mL/min, and the column

162

temperature was kept at 35°C. Detection was at 283 nm for flavanones and at 330 nm for

163

PMFs. The concentration range, LOD, LOQ and linearity of standards were listed in

164

Supplementary Table 2. The structure and UV spectrum of the identified flavonoids were

165

listed in Supplementary Table 3.

166

Fruit storage and sampling. The fruits from CS3 were stored at 16-20°C with RH:

167

80-90%. The flavedo of fruits from 0, 15, 25, 35, 45 days after storage (DAS) was sampled,

168

frozen in liquid nitrogen immediately and kept at –80°C until analysis.

169

Penicillium digitatum inoculation and sampling. Fruits from 35 DAS were used for P.

170

digitatum inoculation according to the previous work.33 Each nine fruits as one

171

independent group were incubated in a storage chamber (25°C, 95% RH). Disease

172

incidence rate and lesion diameter were calculated based on the following equations (2-4):

173

Infection rate of inoculated fruits IRIF, % =

∑ !"# $% &%"'(") %#&(*

174

Infection rate of inoculated fruits IRIF, % =

∑ !"# $% &%"'(") *-$(*

+×


.×

(2) (3)


175 176

Lesion diameter LD, cm =

∑ 2"*&$ )& "("# $% )"'3&4 *-$(* ($(2 !"# $% )"'3&4 *-$(*

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(4)

Two groups were sampled each time at 0, 2, 6, 12, 24, 48, 72 hours after inoculation

177

(HAI). Samples with a radius of 1.5 cm were cut off from the inoculated YP flavedo (IYPF)

178

and inoculated PK flavedo (IPKF) spots, respectively. Samples peeled from the

179

uninoculated YP flavedo (UYPF) and uninoculated PK flavedo (UPKF) spots at 6, 12, 24,

180

48, 72 HAI were used as the control of IYPF and IPKF, respectively. The inoculated and

181

the uninoculated flavedo were cut off from the same fruit. Groups for investigation of

182

pathogenesis process were kept until 240 HAI and also sampled finally. All the samples

183

were immediately frozen in liquid nitrogen, and kept at –80°C until flavonoid analysis.

184

Transmission electron microscopy. Fruits from CS3 without damage were used for

185

transmission electron microscope (TEM) analysis according to a previous method.41 More

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than one hundred individual cells from thirty fields of vision were used to obtain statistics

187

of plastids by Image J.

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RNA isolation and quantitative real time PCR analysis. Total RNA was extracted

189

according to the method of a previous work.31 Integrity of RNA was electrophoretically

190

verified, and then its concentration, A260/A280 and A230/A260 absorption were detected

191

by Nanodrop (Agilent 2100, USA). Potential contamination with DNA was eliminated by

192

treatment with DNase I (RNase-free) (Fermentas MBI). One µg of total RNA from each

193

sample was used to synthesize the first strand cDNA using the RevertAid First Strand

194

cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Waltham, MA), following the

195

manufacturer's recommendations. The qRT-PCR was carried out in an ABI PRISM® 9600

196

Sequence Detection System (Applied Biosystems) using SYBR Green Supermix according

197

to the manufacturer’s instructions, under the thermal cycle conditions of an initial

198

denaturation at 94°C for 10 min, followed by 40 cycles of 94°C for 15 s, 60°C for 31 s for


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annealing, and a final step of extension at 72°C for 7 min. The expression levels of the

200

selected genes were calculated by the delta-delta-Ct method.42 The expression level of

201

β-actin is more stable than that of GPADH, thus the β-actin gene was used as reference

202

gene for data normalizations. Each biological sample was examined in duplicate with three

203

technical replicates. Genes and primers for quantitative reverse transcription-PCR analysis

204

were listed in Supplementary Table 4.

205

Western blot analysis. Total proteins were extracted as described previously,

206

quantified using a RC DC protein assay kit (Bio-Rad, Hercules, CA, USA). Then, 30 ug

207

total flavedo protein or 50 ug total pulp protein was separated by SDS-PAGE (12.5%) and

208

blotted onto PVDF membranes (Millipore, USA). The subsequent western blot analyses

209

were conducted as previously described.41 The primary antibodies (1:3000, v/v) were

210

rabbit anti-plastoglobulin 35 (PGL35) antibody (Agrisera@, Sweden) and mouse anti-plant

211

Actin (as an internal reference, Abbkine@, USA); the secondary antibodies (1:15000, v/v)

212

were peroxidase-conjugated immunopure goat anti-rabbit or goat anti-Mouse IgG [H+L]

213

(Pierce, USA). The signal was detected using a Clarity Western ECL Substrate (Bio-Rad,

214

Hercules, CA, USA) according to the manufacturer’s instructions. The chemiluminescence

215

signal was imaged using a ChemiDoc XRS (Bio-Rad) and quantified using Quantity One

216

software (Bio-Rad, USA). The calculated intensity volumes were fitted with a variable

217

slope dose-response relationship using Image J. The relative value of plastoglobulin 35

218

was calculated by normalization using Actin value.

219

Measurement of H2O2. 0.5 g flavedo powder was homogenized by liquid nitrogen and

220

extracted in 5 mL of physiological saline. Measurement of H2O2 was conducted as

221

previously described.33

222

Statistical analysis. The variance of data was analyzed using SPSS software package


43

and


223

release 16.0 (SPSS Inc. Chicago, IL). Multiple comparisons were performed by One-way

224

ANOVA based on Duncan’s multiple range tests, while paired-samples t-tests were

225

performed to test the statistical significance between two genotypes.

226 227

RESULTS AND DISCUSSION

228

YP is characterized by yellowish coloring with reduced carotenoid and xanthophyll

229

accumulation and better postharvest performance. Yellowish coloring in both the

230

flavedo and pulp was observed in YP at not only ripening stage but also the postharvest

231

stage. No difference in leaf morphology was found between YP and PK (Fig. 1a). The

232

lower CI in YP indicates a slower coloring (Fig. 1b). Compared with that of PK, the total

233

content of carotenoids and xanthophylls in YP was decreased by half in flavedo and by one

234

quarter in pulp (Fig. 1c, top: year 2013, bottom: year 2014). Along with the ripening, TSS

235

showed an increased accumulation while TA showed an obvious decrease in both YP and

236

PK. However, the slower decrease of TA in YP resulted in a less remarkable increase of

237

TSS/TA in YP (Table 1). These results indicate a slight difference of internal quality

238

between YP and PK. We also observed a better postharvest performance of YP, as

239

indicated by a lower decay rate (Fig.S2a) and better quality in YP during storage

240

(Fig.S2b-c). These phenotypes were confirmed to be stable by consecutive investigations

241

for several years.

242

Xanthophyll esters are reduced in YP. Information concerning the natural binding form

243

of carotenoids is usually lost due to the saponification procedure, a commonly used step in

244

carotenoid analysis.20 To obtain a comprehensive understanding of the carotenoid and

245

xanthophyll accumulation process, flavedo samples with and without alkaline-

246

saponification were analyzed (Fig. 2). In unsaponified samples, YP shared the maximum


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wavelengths (440.6 nm and 464 nm) of absorbance with PK (Fig. 2a). In detail, in YP

248

samples, violaxanthin esters and cryptoxanthin esters showed a significantly lower

249

absorbance, but unesterificated carotenoids showed a higher absorbance at each coloring

250

stage (Fig. 2c, left).

251

In the flavedo, the contents of α-carotene, β-carotene, zeaxanthin, and lutein isomers

252

were one fold or more lower in YP than in PK; the content of lutein was one fold lower in

253

the year 2013 but showed no decrease in the year 2014 in YP; violaxanthin and

254

9-cis-violaxanthins in YP showed about one-fold decrease in the year 2013 but only a

255

slight decrease in the year 2014 (Fig. 2c, right; Fig. 3a and Fig. S3a). This inconsistency

256

was speculated to be caused by the varying coloring behaviors in different years (Fig. 1b).

257

It is noteworthy that YP flavedo showed a five-fold lower accumulation of β-cryptoxanthin

258

in both years. Compared with in the pulp of PK, in the pulp of YP, a consistent one-fold

259

decrease in the contents of β-cryptoxanthin, lutein and lutein isomer was observed in both

260

years, while the contents of violaxanthin, neoxanthin and zeaxanthin showed no consistent

261

decrease. In addition, it was noted that the content of β-carotene was stably decreased by

262

four or more folds (Fig. 3b and Fig. S3b).

263

As compared with in violaxanthin-abundant citrus, the main contributor to the

264

orange-reddish appearance in Ponkan pulp is β-cryptoxanthin (violaxanthins and

265

β-cryptoxanthin in flavedo).29 However, unlike both the fruits of violaxanthin-abundant

266

type and fruits of β-cryptoxanthin-abundant type, YP showed steadily reduced contents of

267

total carotenoids and xanthophylls (mainly β-cryptoxanthin) in both the pulp and flavedo.

268

These results indicate different behaviors of carotenoid accumulation and esterification

269

between YP and PK. It is noteworthy that the carotenoid profile of YP was also different to

270

that of Pinalate, a yellowish mutant of Navelate orange (Citrus sinensis L. Osbeck), which



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271

accumulates linear carotenes.31

272

Abscisic acid accumulation is higher in YP at earlier coloring stages. To investigate

273

whether ABA accumulation is influenced by altered carotenoid composition in YP,

274

quantification of ABA was performed in flavedo and pulp (Table 2). In the flavedo, ABA

275

content started to decrease at CS2 in PK but was maintained at higher levels at CS1 and

276

CS2 in YP. In the pulp of both genotypes, ABA content increased from CS1 to CS3.

277

However, YP pulp showed a higher accumulation of ABA at CS1 (about 2-fold increase)

278

and CS3 (38% increase) when compared with PK pulp. In total, YP has a higher

279

accumulation of ABA at the earlier period. Results indicated that the reduced accumulation

280

of xanthophylls showed strongly correlation with the ABA accumulation in YP. This

281

conclusion was supported by the expression of NCED5 in the after-mentioned results (Fig.

282

6).

283

Organic acids and flavonoid are the main differentially accumulated metabolites

284

during coloring. Fifty-two and fifty-seven identified primary metabolites were detected in

285

the pulp and flavedo, respectively, mainly including sugars, organic acids, amino acids,

286

alcohols, fatty acids and several other metabolites (Table 3). All together there were

287

forty-one metabolites in the pulp and forty-eight metabolites in the flavedo had

288

significantly different accumulations between YP and PK. It is noteworthy that the

289

accumulations

290

12-tricosadiynoic acid) in the pulp and isocitric acid in the flavedo were significantly

291

different at all three CSs between YP and PK (p < 0.01). Briefly, the dominant sugars were

292

lower in YP pulp while the dominant organic acids were higher in both the flavedo and

293

pulp of YP when compared with in the flavedo and pulp of PK.

294

of

four

metabolites

(xylose,

ribofuranose,

succinic

acid,

10,

Analyses of secondary metabolites were performed on coloring fruits by LC-MS/MS.


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295

Except for flavonoid, other phenolic acids (protocatechuic acid, p-hydroxybenzoic acid,

296

caffeic acid, vanillic acid, coumaric acid, ferulic acid, sinapic acid) showed no significant

297

difference between YP and PK (data not shown). Five flavanones (eriocitrin, ERI;

298

hesperidin, HES; poncitrin, PON; Narirutin, NART; didymin, DID) and four PMFs

299

(sinensetin, SIN; nobiletin, NOB; sinensetin, SIN; 5-demethylnobiletin, DEM) were

300

indentified (Fig. 4a and Table S2-3) and quantified for the two consecutive years (Fig. 4b

301

and Fig. S4). In flavedo, higher accumulations of ERI, HES and PON were found in YP

302

compared with in PK in the year 2013. Similar accumulations of HES and PON were

303

observed in YP in the year 2014. NART and DID showed no significant difference

304

between YP and PK in the year 2013, while NART showed lower accumulation at CS2 and

305

CS3 in YP in the year 2014. Furthermore, in YP flavedo, all the PMFs at CS2 and CS3

306

were significantly higher in the year 2013, while NOB at CS2, SIN and DEM at CS3

307

showed a significantly higher level of accumulation in year 2014, as compared with in PK

308

flavedo (Fig. 4b and Fig. S4a). In YP pulp, a significantly higher level of HES was found

309

at CS1 in the year 2013 and at CS2 and CS3 in the year 2014. Interestingly, in YP pulp,

310

higher accumulations of PMFs were found at CS1 and CS2 in the year 2013, but at CS1

311

and CS3 in the year 2014 (Fig. S4b-c).

312

The results showed that in addition to some sugars and the dominant organic acids

313

(citric acid, malic acid, succinic acid, isocitric acid), PMFs were the main different

314

metabolites between YP and PK. Due to the anticancer and antivirus activities and other

315

benefits of PMFs to human, the YP fruits might be more attractive as fresh fruit for their

316

higher level of PMFs. Furthermore, higher levels of PMFs mean higher antimicrobial

317

activity in YP flavedo.36

318

YP has enhanced resistance against Penicillium digitatum. P. digitatum was used as an



319

elicitor to test the resistance of fruits from 45 DAS to pathogen infection (Fig. 5). After

320

inoculation, infection lesion appeared on PK fruits at 60 HAI, while no infection lesion

321

was found on YP fruits (Fig. 5a). Inhibited development of P. digitatum and a lower rotting

322

rate were found during the infection process of P. digitatum in all YP groups when

323

compared with in PK groups (Fig. 5a-b). Statistical results of IRIS and IRIF and LD from

324

60 HAI to 144 HAI indicated a higher infection rate in PK than in YP, though the infection

325

rate varied among three groups of the same genotype (Fig. 5c-e). We previously found that

326

YP showed a lower rotting rate and better internal quality in storage experiments for three

327

consecutive years (Fig. S2). We speculated that the antimicrobial activity might be

328

improved in YP flavedo. Here, our results indicated that the development of P. digitatum

329

was more effectively inhibited in YP compared with in PK.

330

The increase in expression of carotenoid catabolism genes exceeds that of biosynthesis

331

genes in YP vs. PK. To understand the regulation of carotenoid accumulation at the

332

transcription level, the relative expression of carotenoid-related genes was investigated

333

(Fig. 6). The dominantly expressed deoxy-D-xylulose 5-phosphate synthase (DXS) gene

334

DXS1 showed significantly lower expression at CS1 and CS2 in YP as compared with in

335

PK. Interestingly, deoxy-D-xylulose 5-phosphate reducto- isomerase (DXR) and

336

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) showed significantly

337

higher expression at all CSs while isopentenyl diphosphate isomerase (IPI) and GGPP

338

synthase (GGPS) showed several-fold higher expression at CS3 in YP relative to PK. In

339

YP pulp, DXS1 and DXS2 showed relatively higher expression at all CSs, while IPI and

340

GGPS showed higher expression at CS3 (Fig. 6).

341

In the flavedo, most of the carotenoid biosynthesis genes except for ζ-carotene

342

desaturase (ZDS) showed higher expression during coloring especially at CS3 in YP than


Page 16 of 48

Page 17 of 48


343

in PK (Fig. 6a). In the pulp, most of these genes showed decreased expression at CS1 but

344

no different expression or only slightly increased expression at the last two CSs in YP

345

relative to PK (Fig. 6). It is noteworthy that the dominantly expressed phytoene synthase

346

(PSY) gene, PSY1, showed 12-fold expression in YP flavedo and 3-fold expression in YP

347

pulp at CS3 compared with in PK flavedo and pulp (Fig. 6).

348

The up-regulation of the expression of most of the genes for ABA synthesis and

349

carotenoid cleavage was stronger than that of the genes of MEP pathway and carotenoid

350

biosynthesis at the later CSs in YP (Fig. 6). The expression of 9-cis-epoxy-carotenoid

351

dioxygenase (NCED) 3 was stable and showed no significant difference between YP and

352

PK, while NCED5 showed more than twenty-fold higher expression in YP flavedo at all

353

three CSs and three-fold expression in YP pulp at CS3 when compared with its expression

354

in PK. In addition, the expression of CCD1 and CCD4b1 was two or three folds higher at

355

all CSs in YP flavedo, while CCD1 showed no difference in expression between YP and

356

PK in the pulp. Surprisingly, CCD4b1 was only detected in YP pulp. However, compared

357

with in PK pulp, more than five-fold higher expression of CCD4b2 was found in YP pulp

358

at CS2 and in YP flavedo at CS3, and more than seven-fold higher expression of CCD4c

359

was observed in YP pulp at CS3 in the present study (Fig. 6).

360

NCED5 and NCED3 cleave 9-cis-violaxanthin at the 11-12 position to form xanthoxin,

361

a precursor of ABA, while CitCCD1 protein cleaves β-cryptoxanthin, zeaxanthin, and

362

all-trans-violaxanthin at the 9-10 and 9’-10’ positions and 9-cis-violaxanthin at the 9’-10’

363

position.26 The twenty-fold higher expression of NCED5 might result in the higher

364

accumulation of ABA in YP flavedo (Table 2). It is worthy to note that CCD4b1 in citrus

365

(CitCCD4) was proved to produce a fruit-specific C30 apocarotenoid (β-citraurin) from

366

cryptoxanthin and zeaxanthin.27-28 However, β-citraurin was not detected in both YP and



367

PK. Thus, except for the production of β-citraurin, CCD4b1 might be involved in other

368

catalytic reactions. CCD4c is restricted to petals and may be related to the cleavage of

369

carotenoids to colourless volatile apocarotenoids (C9 to C13). CCD4a transcripts were

370

detected in all of pulp, peel, leaf, stem, root and petal but were relatively higher in leaf.28

371

For their unclarified functions and low absolute expression levels, the relationship between

372

CCD4a, CCD4b2 or CCD4c and carotenoid catabolism in citrus fruits has to be further

373

investigated.

374

Although the expression of upstream genes (DXR, HDS, IPI, GGPS and PSY) was

375

unexpectedly up-regulated in YP, the reduced carotenoid accumulation of YP might be due

376

to that the increase in expression of carotenoid catabolism genes exceeds that of

377

biosynthesis genes in YP vs. PK. The unexpected up-regulation of upstream genes for

378

carotenoid biosynthesis in YP at CS3 might be due to the feedback regulation by the higher

379

accumulation of ABA at the earlier coloring stages and a potential higher oxidative stress

380

as indicated by the higher H2O2 level (Fig.S8). Furthermore, it was recently reported that

381

OR controls carotenoid biosynthesis via posttranscriptional regulation of PSY in plants.18

382

Thus, the transcripts of these up-regulated genes may not always be translated to active

383

proteins because of the potential regulation at posttranscriptional level.

384

Carotenoid sequestration capacity and compartment of chromoplastid are reduced in

385

YP. Western blot analysis was performed to detect fibrillin/PGL35 protein, a lipoprotein in

386

plastoglobulus related to lipid storage and carotenoid and tocopherol synthesis.17

387

Compared with in PK flavedo and pulp, significant lower abundance of PGL35 at CS1 and

388

CS2 was found in YP flavedo (Fig. 7a), while lower abundance of PGL35 at CS1 was

389

found in YP pulp (Fig. 7b).

390

As shown in Fig. S5a, to investigate the compartment of chromoplastid in flavedo,


Page 18 of 48

Page 19 of 48


391

more than 100 cells from 30 TEM sections were used to obtain statistics for

392

chromoplastids. The average number and density of plastids were lower in YP flavedo than

393

in PK flavedo: 38.78% of the investigated YP cells held one plastid, which is obviously

394

higher than that of PK (20.69%); while only 3.06% of YP cells held four chromoplastids,

395

which is far less than that of PK (15.86%) (Fig. S5b). In addition, more plastids of 0.00 to

396

7.00 um2 while less plastids of 7.01 to 16.00 um2 were found in YP compared with in PK

397

(Fig. S5c). Similarly, more cells with 0.00 to 10.00% total plastid compartment area

398

(TPCA) while fewer cells with 12.00 to over 30.00% TPCA were present in YP relative to

399

in PK (Fig. S5d). In summary, compared with those from PK, plastids from YP were

400

characterized by lower density, smaller size and smaller TPCA. These results indicate

401

reduced storage capacity for carotenoids and xanthophylls in YP.

402

The relatively lower abundance of PGL35 in YP fruits at earlier stages indicates

403

reduced capacity of carotenoid sequestration during coloring. Recently, the QTL with the

404

largest effect on β-cryptoxanthin content was detected. This QTL includes the Gn0005

405

locus, a marker derived from the CitPAP cDNA sequence (Acc. No. AB011797).44

406

Although the relation between CitPAP and carotenoid accumulation has not been clarified

407

in citrus, the reduced abundance of PGL35 in YP at the earlier stages of coloring showed a

408

strongly positive correlation with the reduced carotenoid accumulation. Thus, our results

409

suggest that the carotenoid accumulation in YP is the integrated result of the

410

disequilibrium between carotenoid catabolism and biosynthesis and the weakened storage

411

capacity of carotenoids.

412

PMFs levels are stabilized and H2O2 accumulation is increased in YP during

413

Penicillium digitatum expansion. As shown in Fig. S6, compared with in PK, PAL1

414

expression was up-regulated in YP flavedo but was not up-regulated in YP pulp during



415

coloring. However, the accumulation of PMFs was increased in both of the flavedo and

416

pulp of YP when compared with that in PK (Fig. 4b and Fig. S4). Thus, the correlation

417

between the expression of PAL1 and PMFs accumulation might be tissue-specific because

418

flavedo is the interface to environment and is subjected to virous stresses. In addition, the

419

expression of PAL1 showed positive correlation with the abscisic acid content in flavedo.

420

An ABRE cis-element was found in the promoter of PAL1. Thus, we speculated that the

421

increased accumulation of ABA might increase the flavonoid accumulation by

422

up-regulating the expression of PAL1.

423

During 45 days postharvest storage, the levels of PMFs fluctuated and reached the

424

highest value at 15 DAS, but were totally significantly higher in YP flavedo compared

425

with in PK flavedo, except for NOB, DEM and TAN (tangeretin) at 35 DAS (Fig. S7).

426

Thus, higher levels of PMFs in YP at both coloring and storage stages might be a

427

biochemistry basis for the enhanced resistance. Consistent with this deduction, the YP

428

fruits from 45 DAS showed enhanced resistance against P. digitatum (Fig. 5).

429

To understand the biochemistry basis for the enhanced resistance against P. digitatum,

430

a sequential monitoring of secondary metabolites was conducted in inoculated or

431

uninoculated flavedo by UPLC (Fig. 8) and LC-MS/MS. In previously reported studies, the

432

inoculated and uninoculated materials for monitoring flavonoids were sampled from

433

different fruits.34,

434

uninoculated flavedo materials for flavonoid detection from the same fruit. Since the same

435

fruit means the same physiological background, our sampling method should be more

436

reliable. No novel metabolite derived from the inoculation was found (data not shown).

437

The contents of all the flavonones fluctuated but in general showed higher levels in YP

438

(IYPF/UYPF) relative to PK (IPKF/UPKF) during 240 HAI. HES’ was deduced to be

37-38, 45

Different to these methods, we sampled the inoculated and


Page 20 of 48

Page 21 of 48


439

Hesperetin 7-o-glucoside in previous document, which is the probable product of HES

440

hydrolysis by enzymes from fungi.45 It is noteworthy that the levels of HES’ and HES’’

441

(another unknown flavonone) were increased by folds in YP than in PK at 240 HAI. The

442

contents of PMFs in IPKF and UPKF did not increase and started to decrease from 12 HAI

443

and 24 HAI, respectively; while their contents in IYPF and UYPF were increased and

444

reached peak values at 6 HAI and 12 HAI, respectively. It is noteworthy that the PMFs in

445

UYPF stayed unchanged from 12 HAI to 72 HAI and started to decrease slowly afterward.

446

Our results indicate that the stabilized level of PMFs in the uninoculated flavedo in YP

447

help to construct an effective barrier against the attack of P. digitatum.

448

H2O2 was proved to be an important secondary messenger for the induction of the

449

expression of defense genes.46 It is noteworthy that H2O2 level was higher in both

450

inoculated and uninoculated spots in YP flavedo than that in PK flavedo during P.

451

digitatum expansion (Fig. S8). The integration of reactive oxygen species/redox status and

452

sugars/carbon status can account for most of the effects of the major environmental factors

453

that influence carotenoid biosynthesis, and carotenoids or their derivatives can in turn act

454

as stress signaling molecules.47 Reasonably, H2O2 might participate in the regulation of

455

carotenoid biosynthesis and the induction of the expression of defense-related genes. A

456

more efficient transduction of the H2O2 signal from the inoculated spots to healthy tissues

457

may facilitate the establishment of barriers against pathogens, such as the accumulation of

458

PMFs in YP.

459

The volatile oil in flavedo is another group of important metabolites, which might be

460

related to the resistance against Penicillium digitatum.35, 48 We conducted a comparative

461

analysis of chemical composition of the volatile oil in flavedo between YP and PK. As the

462

previously proved anti-fungal components against Penicillium digitatum, D-limonene,



463

γ-terpinene and β-linalool showed lower accumulation, while α-Terpineol and Octanal

464

showed higher accumulation in YP flavedo when compared with in PK flavedo. (Table S5;

465

Figure S9). However, the anti-fungal activity of α-Terpineol and Octanal against

466

Penicillium digitatum might be weak due to their low accumulation in flavedo (Table S5).

467

In total, the content of anti-fungal components was lower in YP than in PK. Thus, volatile

468

oil might not contribute to the enhanced resistance in YP.

469

In conclusion, our results demonstrate that the disequilibrium between catabolism and

470

biosynthesis and the reduced sink capacity of carotenoids are responsible for the reduced

471

xanthophylls and the yellowish coloring in YP fruits. Through a comprehensive

472

investigation of YP by analyzing its primary and secondary metabolites and resistance

473

against abiotic and biotic stresses, this study reveals that reprogramming of metabolisms

474

may help to enhance the resistance against abiotic and biotic stresses in YP. However, the

475

improvement of postharvest performance was at the expense of a part of nutritional quality

476

such as carotenoids, while higher PMFs might compensate for this loss of nutrition in the

477

pulp. Taken together, our work is the first report that confirms the practicability of using

478

the reprogramming of metabolisms in mutants to improve the postharvest performance of

479

citrus fruit.

480 481

ACKNOWLEDGEMENT

482

We are grateful to Prof. Zuoxiong Liu (Foreign Language College of Huazhong

483

Agricultural University) for reading and revising the manuscript.

484 485

FUNDING

486

This work was supported by Huazhong Agricultural University Scientific & Technological


Page 22 of 48

Page 23 of 48


487

Self-innovation Foundation, the National Natural Science Foundation of China (NSFC,

488

Grant nos. 31221062 and 31271968), the Program for New Century Excellent Talents in

489

University (NCET-12-0859), the National Modern Agriculture (Citrus) Technology

490

Systems of China (Grant nos. CARS-27) and the Special Fund for Agro-scientific

491

Research in the Public Interest (Grant nos. 201303093).

492 493

CONFLICT DECLARATION

494

All authors declare that they have no conflict of interest.

495 496

SUPPORTING INFORMATION DESCRIPTION

497

Supplementary Table 1. Retention time and UV spectrum of isolated carotenoids in

498

HPLC analysis;

499

Supplementary Table 2 Structure and UV spectrum information of identified flavonoids

500

by UPLC analysis;

501

Supplementary Table 3 Data for calibration curve of thirteen flavonoid standards;

502

Supplementary Table 4 Specific primers used in real-time reverse transcriptase-PCR;

503

Supplementary Table 5 Chemical composition and relative content of the volatile oil from

504

flavedo of YP and PK.

505

Supplementary Figure 1. Pathway for carotenoid biosynthesis and cleavage;

506

Supplementary Figure 2. Changes in rotting rate and internal quality during postharvest

507

storage.

508

Supplementary Figure 3. Carotenoid contents from fruits of year 2014 at three coloring

509

stages;

510

Supplementary Figure 4. Compositions and contents of flavonoids in fruits at coloring



511

stages.

512

Supplementary Figure 5. Statistics of plastids according to transmission electron

513

microscope of flavedo.

514

Supplementary Figure 6. Expression of PAL1 in flavedo and pulp in both genotypes.

515

Supplementary Figure 7. Changes in compositions and contents of flavonoids in flavedo

516

during 45 day storage.

517

Supplementary Figure 8. H2O2 contents in flavedo of PK and YP during P. digitatum

518

infection.

519

Supplementary Figure 9. GC-MS analysis of chemical composition of the volatile oil

520

from flavedo of PK and YP.

521

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

522


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523 524

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629

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Figure captions

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Figure 1. Phenotype of Ponkan and its mutant. (a) Coloring and shape of leaves and

687

fruits. Top part: leaves and fruits were harvested on tree; bottom part: the whole fruits,

688

pulp (juice sac) and flavedo were from 20 DAS. (b) CI of PK and YP at three coloring

689

stages for two consecutive years. (c) Total content of carotenoids and xanthophylls in

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flavedo and pulp at three coloring stages. Top part: year 2013, bottom part: year 2014.

691

Error bars represent SDs of the means (n ≥ 3, biological triplicates). Results were averaged

692

value plus SDs of the means and statistically analyzed using a paired-samples t-test. *:

693

significant difference, p

Distinct Carotenoid and Flavonoid Accumulation in a Spontaneous

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