Peach carboxylesterase PpCXE1 is associated with catabolism of

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Chemistry and Biology of Aroma and Taste

Peach carboxylesterase PpCXE1 is associated with catabolism of volatile esters Xiangmei Cao, Kaili Xie, Wenyi Duan, Yunqi Zhu, Mingchun Liu, Kunsong Chen, Harry J Klee, and Bo Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01166 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Journal of Agricultural and Food Chemistry

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Peach carboxylesterase PpCXE1 is associated with catabolism of volatile esters

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Xiangmei Cao1, Kaili Xie1, Wenyi Duan1, Yunqi Zhu2, Mingchun Liu2, Kunsong

3

Chen1, Harry Klee1,3, Bo Zhang 1,*

4

1Laboratory

5

Horticultural Plant Integrative Biology, Zhejiang University, Zijingang Campus,

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Hangzhou 310058, China

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2Key

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College of Life Sciences, Sichuan University, Chengdu 610065, China

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3Horticultural

10

of Fruit Quality Biology/Zhejiang Provincial Key Laboratory of

Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,

Sciences, Plant Innovation Center, Genetic Institute, University of

Florida, Gainesville, FL 32611, USA

11 12

Corresponding Author: Bo Zhang

13

Tel: +86-571-88982471

14

Fax: +86-571-88982224

15

E-mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract

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Peach fruit volatile acetate esters impact consumer sensory preference and contribute

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to defense against biotic stresses. Previous studies showed that alcohol acyltransferase

19

(AAT) family PpAAT1 is correlated with volatile ester formation in peach fruits.

20

However, fruits also contain carboxylesterase (CXE) enzymes that hydrolyze esters.

21

The functions of this family with regard to volatile ester content has not been explored.

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Here, we observed that content of acetate ester was negatively correlated with

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expression of PpCXE1. Recombinant PpCXE1 protein exhibited hydrolytic activity

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toward acetate esters present in peach fruit. Kinetic analysis showed that PpCXE1

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showed the highest catalytic activity to E-2-hexenyl acetate. Subcellular localization

26

demonstrated that PpCXE1 is present in the cytoplasm. Transient expression in peach

27

fruit and stable overexpression in tomato fruit resulted in significant reduction of

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volatile esters in vivo. Taken together, the results indicate that PpCXE1 expression is

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associated with catabolism of volatile acetate esters in peach fruit.

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Keywords: CXE, flavor, fruit, volatile esters

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Introduction

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Consumers are increasingly dissatisfied with the flavor of many horticultural crops,

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including tomato, strawberry and peach fruit. Flavor is an elusive quality trait due to

35

its inherent complexity. Many volatile and non-volatile chemicals contribute to

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overall flavor and it is highly influenced by environment and agricultural practices.

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As a consequence of that complexity, breeding programs have not systematically

38

focused on flavor, instead of focusing efforts on yield, disease resistance and

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postharvest handling traits.1 Volatile esters are important compounds that contribute

40

to the characteristic aroma of many fruits. Sensory analysis indicated that volatile

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acetate esters such as Z-3-hexenyl acetate are positively correlated with consumer

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liking of peach fruit.2 In addition to their contributions to flavor, volatile esters are

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involved in plant defense against biotic stresses. Reduced production of volatile esters

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in tomato, for example, affected stomatal closure and led to hyper-susceptibility to

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infection with Pseudomonas syringae pv. Tomato.3 For peach fruit, a high content of

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volatile esters such as Z-3-hexenyl acetate was suggested to enhance resistance to

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Mediterranean fruit fly Ceratitis capitata.4

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Overall fruit volatile ester content is the consequence of a balance between

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anabolism and catabolism. They are synthesized by alcohol acyltransferases (AATs)

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that transfer an acyl moiety from acyl-coenzyme A donors to alcohol acceptors,5 and

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broken down by carboxylesterase (CXE) activity. Multiple members of AAT gene

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families have been identified in fruits, including tomato,6 apple,7,8 melon,9

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strawberry,10 kiwifruit11 and peach.12,13 Much less is known about catabolism of

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volatile esters in fruit.

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CXEs (EC 3.1.1.1) belong to the α/β hydrolase superfamily.14-16 Plant CXEs are

56

involved in regulating biological activity and transmembrane transport of natural 3



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products,17,18 bioactivation of herbicides,19,20 detoxification and pathogen defense.21-24

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Tobacco SABP2 (SA-binding protein) and tomato MJE (methyl jasmonate esterase)

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are responsible for hydrolysis of methyl salicylate to salicylic acid and methyl

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jasmonate to jasmonic acid, respectively.25,26 Twenty CXE gene family members have

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been identified in Arabidopsis (AtCXE1 to AtCXE20).14 Some work has been

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performed on CXE enzymes in fruit systems. As in Arabidopsis, apple contains a

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large gene family where MdCXE1 is associated with hydrolysis of acetate esters.27

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The role of CXE proteins in regulating volatile ester content has been most

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extensively studied in tomato (Solanum lycopersicum) where one enzyme, SlCXE1

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has a major role in determining volatile acetate ester content.

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CXE is the conserved catalytic triad, whose active site is made up of a serine

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(surrounded by sequence GXSXG), a glutamate and a histidine. Another structural

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motif of importance is the oxyanion hole (HGG), which is involved in stabilizing the

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substrate-enzyme intermediate during hydrolysis.14

28

The main feature of

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Distinct profiles of volatile esters have been observed between different peach

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varieties,29,30 and contents of fruit esters are also affected by postharvest

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treatments.12,31 In this study, we examined whether any member(s) of the peach CXE

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gene family are associated with volatile acetate ester contents in different peach

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varieties or in response to postharvest fruit treatments. In vitro enzymatic assays

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indicated that PpCXE1 hydrolyzes the acetate esters detected in peach fruit. Transient

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overexpression in peach fruit and stably transformed tomato fruit were used to

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demonstrate that PpCXE1 is responsible for hydrolysis of aromatic esters in planta.

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Together, the data support a model in which PpCXE1 has a major role in controlling

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volatile acetate esters in peach fruit.

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Materials and Methods

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Plant materials and treatments

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Fruits from five cultivated peach varieties including Prunus persica L. Batsch cv.

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LaoMiTao (LMT), DaYeZiMi (DYZM), HanLuMi (HLM), HuJingMiLu (HJML),

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ZaoShangHaiShuiMi (ZSHSM) were harvested at commercial maturity from the

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Melting Peach Research Institute of Fenghua (Zhejiang Province, China). Fruits from

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three wild peach varieties including P. kansuensis Rehd. cv. HongGenGanSuTao

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(HGGST),

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ZhouXingShanTao (ZXST) were harvested from Zhengzhou Fruit Research Institute

90

(Henan Province, China) (Supplementary Figure S1). HJML peach fruits were soaked

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with 1 mM MeJA solution32 for 10 min. Control fruits were treated with water for 10

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min. After treatment, fruits were stored at 0o C for 0 d, 21 d and 28 d followed by

93

three days shelf-life at 20o C (+3 d), respectively. Fruit mesocarp (~5 mm) were

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separated and frozen in liquid nitrogen, and then kept at -80

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irradiation treatment was applied to delay ester accumulation of peach fruit according

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to our previous study.33 HJML peach fruits were divided into two groups: one group

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was exposed to 150 μW cm-2 UV-B (280~315 nm) up to 48 h in a climatic chamber

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with 20o C and 90 % relative humidity. The control group was covered with aluminum

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foil and placed in a same climatic chamber. Fruits were sampled after 6 h and 48 h

100

treatment and exocarp (~1 mm) were separated and frozen in liquid nitrogen, then

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kept at -80o C until use. Three biological replicates of five fruits each were used at

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each sampling time point.

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Volatile collection and GC-MS analysis

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Fruit volatile compounds were analyzed according to the method described

105

previously.31 After ground into powder in liquid nitrogen, 5 g of frozen flesh tissues

BaiGenGanSuTao

(BGGST)

and

P.

5


davidiana

o

Franch.

cv.

C until use. UV-B


transferred

to

20

mL

vials

containing

3

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were

mL

of

200

mM

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ethylenediaminetetraacetic acid (EDTA) and 3 mL of 20 % CaCl2. Before the vials

108

were sealed, 30 μl of 2-octanol (0.8 mg mL-1) was added as an internal standard. A

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fiber coated with 65 μm of polydimethylsiloxane and divinylbenzene (PDMS-DVB)

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(Supeclo Co., Bellefonte, PA, USA) was used for volatile collection. The vials were

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placed in the tray of a solid-phase micro extraction (SPME) autosampler (Combi PAL,

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CTC Analytics, Aligent Technologies, USA), coupled to an Agilent 7890N gas

113

chromatograph and an Agilent 5975C mass spectrometer.

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Volatile esters were separated on a DB-WAX column (30 m × 0.25 mm i.d. ×

115

0.25 μm film thickness; J & W Scientific, Folsom, CA, USA). The temperature

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program started at 40o C and was increased by 3o C min-1 to 100o C, and then to 245o C

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at 5o C min-1. Helium was employed as carrier gas with a flow rate of 1.0 mL min-1.

118

The column effluent was ionized by electron ionization (EI) at energy of 70 eV with a

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transfer temperature of 250o C and source temperature of 230o C. Volatiles were

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identified by comparison of the mass spectra of the NIST Mass Spectral Library

121

(NIST-08) and retention times of authentic standards. Quantification of compounds

122

was performed using the peak area of the internal standard as a reference and response

123

factors based on total ion chromatogram (TIC). The retention index and response

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factor calculation of volatiles by GC-MS was listed in Table S1.

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Gene expression analysis

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Total RNA was extracted according to the method described by Zhang et al.,34 then

127

treated with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa) to remove

128

possible genomic DNA contamination and synthesize the first strand cDNA. Gene

129

expression was analyzed by RNA-sequencing (RNA-Seq) and real-time quantitative

130

PCR (qPCR). The libraries for high-throughput Illumina strand-specific RNA-seq

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were prepared as previously described.35 Three biological replicates for each ripening

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stage and treatment were performed. qPCR was carried out with a Ssofast Eva Green

133

Supermix Kit using a CFX96 instrument (Bio-Rad). The temperature program for

134

qPCR was as follows: 95o C for 3 min, 45 cycles of 95o C for 10 s and 60o C for 30 s,

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with a final melting curve step from 65o C to 95o C. No-template controls were

136

included in each run. At least three different RNA isolations and cDNA syntheses

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were used as templates for real-time quantitative PCR analysis. The specificity of

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primers used for qPCR analysis was confirmed by product sequencing and listed in

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Table S2.

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Gene sequence analysis

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CXE genes were identified by a BLAST search of the peach genome

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(https://phytozome.jgi.doe.gov) using tomato SlCXE1 and apple MdCXE1 as query

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sequences. Multiple sequence alignments of full-length predicted amino acid

144

sequences were performed using Clustal W with default parameters. The phylogenetic

145

analysis was constructed by neighbor-joining methods by MEGA 6.0 with the matrix

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of the evolutionary distances calculated by Poisson correction for the multiple

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substitutions. The reliability of the tree was evaluated by bootstrap test with 1000

148

replicates. The GenBank accession numbers for CXEs are: SlCXE1 (NP_001307232),

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MdCXE1 (XP_008382301), AtCXE1 (NP_173353), AtCXE12 (NP_190438),

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AtCXE13

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CXEs was calculated by DNAMAN (V8.0). Full length cDNAs of PpAAT1 and

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PpCXE1 in different peach varieties were cloned using the primer in Table S3 and

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cloned to pGEM-Teasy vector for sequence homology analysis.

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Heterologous protein expression and purification

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Recombinant protein purification was performed following the method described

(NP_190439) and AtCXE18 (NP_197744). The sequence homology of

7



156

previously.33 The full-length cDNA of the PpCXE1 gene was inserted into the

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pET-6×HN expression vector (Clontech, Mountain View, CA) with an N-terminal

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His-tag using primers in Table S3. After sequence validation, the construct was

159

transformed into E. coli BL21(DE3) pLysS (Promega, Madison, WI, USA). The

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transformed cells were precultured overnight at 37o C in Luria-Bertani (LB) medium

161

containing 100 μg mL-1 ampicillin. The cultures were grown at 37o C at 180 rpm until

162

the optical density at 600 nm reached 0.6 to 0.8. After cooling the culture to 16o C,

163

isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1

164

mM to induce protein expression. Cultures were incubated overnight at 16o C and

165

were harvested by centrifugation (6000 g, 4o C, 10 min), then resuspended in Tris-HCl

166

buffer (100 mM Tris, 2 mM dithiothreitol, pH 7.0). Negative controls consisted of E.

167

coli. BL21(DE3) pLysS containing the empty expression vector. The resuspended

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cells were disrupted by freeze-thaw, and the crude protein was extracted by

169

centrifugation (12000 g, 4o C, 30 min), followed by filtration with a 0.45 μm filter

170

(Millipore). Recombinant proteins were purified by His TALON gravity column

171

(Clontech) following the manufacturer’s instructions. The presence of the expressed

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proteins was confirmed by SDS-PAGE.

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Enzyme activity assay

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Enzyme activity assays were performed in 500 μl reaction mixture containing

175

Tris-HCl buffer (100 mM Tris, pH 7.5, 2 mM dithiothreitol), 20 μl ester substrates (1

176

mM hexyl acetate, E-2-hexenyl acetate and Z-3-hexenyl acetate, respectively) and 10

177

μl purified protein (1 μg μl-1). The reaction mixtures were incubated at 30o C for 30

178

min. The products were detected by GC-MS. The optimum enzyme reaction

179

conditions were determined with Z-3-hexenyl acetate as substrate under different pH

180

(6.5, 7.0, 7.5, 8.0, 8.5, 9.0) and temperature (25, 30, 35, 40, 45, 50o C) conditions.

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Enzyme kinetics assays were performed in 200 μl reaction mixture, containing

182

Tris-HCl buffer (100 mM Tris, pH 8.0, 2 mM dithiothreitol), ester substrates with

183

different concentration (0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5 mM) and 1 nmol purified protein.

184

The reactions were performed at 35o C for 10 min, then stopped by adding 1 μl of 24

185

% (v/v) TCA. The products were extracted by 100 μl dichloromethane, 1 μl

186

dichloromethane extracting solution was analyzed by GC-MS. The contents of

187

products were calculated with standard curves. The Km values were calculated using

188

non-linear regression analysis of initial rate data with Origin Pro 8 (OriginLab, USA).

189

Kcat values were calculated from Vmax.

190

Transient overexpression in peach fruit

191

Transient overexpression was carried out in peach fruits to verify gene function in

192

vivo according to the method described previously.31 A full length CDs of PpCXE1

193

was cloned into pGreen 002962 SK vector (EU048865) using the primers listed in

194

Table S3. Constructs were electroporated into Agrobacterium tumefaciens GV3101

195

with infiltration buffer (10 mM MES, 10 mM MgCl2, 150 mM acetosyringone, pH

196

5.6). Peach fruits were soaked in sodium hypochlorite solution for 20 min to sterilize

197

and washed three times with sterile water. Four slices of flesh were taken from

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opposite sides of each fruit, two were infected by A. tumefaciens carrying the

199

PpCXE1 construct and the other two were infected by A. tumefaciens carrying the

200

empty construct as negative control. The flesh slices were submerged in A.

201

tumefaciens suspension and subjected to a vacuum (-70 kPa). The vacuum was

202

released slowly to assist bacterial invasion into the flesh tissue. After vacuum

203

infiltration, the flesh slices were rinsed three times with sterile water and cultured on

204

MS medium in a growth chamber (20° C, RH 85 %). After three days, flesh slices

205

were sampled for volatiles by GC-MS analysis. Transient expression assays were

9



206

repeated three times with five fruits each.

207

Stable overexpression in tomato fruit

208

A full length CDs of PpCXE1 was cloned into pBI121 vector (AF485783) using

209

primers listed in Table S3. A. tumefaciens-mediated transformation of tomato (cv

210

MicroTom) plants was carried out according to Wang et al.,36 and transformed lines

211

were selected on a kanamycin-containing (70 mg L-1) medium. Tomato plants were

212

transferred to soil and grown under standard glasshouse conditions. Transgenic and

213

wild type (WT) plants were grown in a greenhouse at 25o C and 16 h light / 8 h

214

darkness. Three plants of each line were sampled for further analysis. Tomato fruit at

215

red ripe stage (breaker +7 days) were harvested for gene expression and ester content

216

analysis. Three biological replicates, each consisting of five fruit, for each line as well

217

as WT were analyzed. Whole fruits were frozen in liquid nitrogen and stored at -80o C

218

until use. The expression of PpCXE1 in transgenic tomato was analyzed by qPCR

219

with primers listed in Table S1.

220

Subcellular localization analysis

221

The recombinant 35S-PpCXE1-GFP vectors were constructed using primers listed in

222

Table S3. The construct was electroporated into A. tumefaciens GV3101. For

223

infiltration, bacteria carrying the target gene were grown in infiltration buffer (10 mM

224

MES, 10 mM MgCl2, 150 mM acetosyringone, pH 5.6) to an OD600 of 0.8~1.0. The

225

vector was infiltrated into four-week-old Nicotiana benthaminana leaves. After 48 h,

226

leaves were detached and observed using a confocal laser scanning microscope (LSM

227

780, Carl Zeiss, Oberkochen, Germany).

228

Statistical analysis

229

Figures were generated by Origin Pro 8 (OriginLab Corporation., Northampton, MA,

230

USA). The two-sample significance test was calculated using unpaired Student's t-test

10


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and least significant difference (LSD) of one-way analysis of variance (ANOVA) was

232

used to test the significance level of multiple groups at significant level (* P < 0.05,

233

** P < 0.01) (SPSS 19.0, SPSS Inc., Chicago, IL).

234

Chemicals and reagents

235

The reference compounds used for volatile identification and enzyme activity such as

236

hexanol, E-2-hexenol, Z-3-hexenol and 2-octanol, hexyl acetate, E-2-hexenyl acetate

237

and Z-3-hexenyl acetate were purchased from Sigma-Aldrich.

238

Results

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Volatile esters in different peach varieties

240

Volatiles derived from fatty acids are the most abundant components in ripe peach

241

fruits (Supplementary Figure S2). The highest proportion of fatty acid-derived

242

volatiles was observed in fruit of P. kansuensis. Major volatiles derived from fatty

243

acids include C6 aldehydes, alcohols, lactones and esters. Great variation of ester

244

content was observed between peach varieties (Supplementary Figure S3). These

245

peach samples could be divided into three groups based on ester content: low (L),

246

medium (M) and high (H). Ester contents of P. persica species LMT and DYZM

247

belong to the L group, with ester content being less than 10 % of total fatty acid

248

volatiles. Wild peach species P. kansuensis and P. davidiana belong to the H group,

249

with more than 30 % of the fatty acid-derived volatiles being esters (Supplementary

250

Figure S3). Content of esters in the H group ranged from 700 to 900 ng g-1 FW,

251

approximately 23-fold higher than the L group (Figure 1A).

252

We next tested if variation in ester content between peach varieties was caused

253

by differences in substrate alcohol contents or transcript levels of PpAAT1 associated

254

with ester biosynthesis. No significant correlation was observed between content of

255

substrate C6 alcohols and corresponding product esters (R=0.05, P=0.80) (Figure 1B).

11



256

Moreover, high content of esters was not correlated with transcript levels of PpAAT1

257

for peach fruits (R=-0.62) (Figure 1C). These results indicated that the contents of

258

substrate alcohols and expression level of PpAAT1 did not explain the variation of

259

ester content between peach varieties. These results prompted us to investigate a

260

potential role for a CXE in ester catabolism in peach fruit.

261

PpCXE1 expression correlates with ester production in peach fruit

262

BLAST searches of the peach genome database using tomato SlCXE1 and apple

263

MdCXE1 peptides as query sequences were performed (Supplementary Figure S4).

264

Levels of transcripts from the PpCXEs in ripe HJML peach fruit were analyzed using

265

RNA-seq (Supplementary Figure S5). The three members (PpCXE1-3) with the most

266

abundant transcripts in peach fruit were cloned for further function analysis. The

267

highest identity of the deduced amino acid sequences was observed between PpCXE1

268

and PpCXE3 (66.1 %), while the lowest identity (53.1 %) was detected between

269

PpCXE2 and PpCXE3 (Supplementary Table S4).

270

Expression analysis showed different abundances of PpCXEs between peach

271

varieties. PpCXE1 had the highest transcript abundance in the L group varieties and

272

lowest abundance in H group peach varieties. A significant negative correlation

273

(R=-0.91, P

Peach carboxylesterase PpCXE1 is associated with catabolism of

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