Phenotypic profiling and gene expression analyses for aromatic and volatile compounds in Chamoes (Cucumis melo)

Page 1

Phenotypic profiling and gene expression analyses for aromaticand volatile compounds in Chamoes (Cucumis melo)

Jeongyeo Lee • Min Keun Kim • Seung Hwan Hwang •

Jungeun Kim • Jong Moon Ahn • Sung Ran Min •

Sang Un Park • Soon Sung Lim • HyeRan Kim

Received: 21 May 2013 / Accepted: 27 January 2014 / Published online: 11 February 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Gotgam chamoe (GgC), a native oriental melon

in Korea, is known to possess the aroma of a dried per-

simmon, an agronomic relevance for melon breeding pro-

gram. The volatile compounds and the transcript levels of

aromatic compound genes in cultivar (Ohbokggul chamoe

[OC]) and GgC were profiled. A total of 62 volatile com-

pounds were identified and quantified. Twenty-eight vola-

tile compounds were specific to either the OC or the GgC.

The amounts of volatile alcohol, saturated hydrocarbon,

and unsaturated hydrocarbon compounds were 2.2, 2.7, and

1.1 times higher in OC, respectively. The amounts of

ketone volatiles were 1.2 times higher in GgC, whereas the

total amounts of esters were similar. In the shikimate

pathway, transcriptional patterns with the fruit parts were

different between the two chamoes for CmDAHPS,

CmDHD/SDH, and CmEPSPS. The expression levels of all

six genes investigated, especially CmCS, were highest in

the peel of both chamoes compared to the other parts. The

transcript levels of the aromatic amino acid biosynthesis

genes demonstrate that phenylalanine and tyrosine are

present more in edible parts of the chamoe, while trypto-

phan may be accumulated low in the chamoe. In addition,

phenylalanine and tryptophan are synthesized more in GgC

than the OC.

Keywords Cucumis melo � Chamoe � Volatile aromatic

compounds � Shikimate pathway � Aromatic amino acids

Abbreviations

Cm Cucumis melo

PE Peel

PU Pulp

S Stalk

OC Ohbokggul chamoe

GgC Gotgam chamoe

GC Gas chromatography

E4P Erythrose 4-phosphate

PEP Phosphoenolpyruvate

Jeongyeo Lee and Min Keun Kim have contributed equally to this

work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-014-3211-9) contains supplementarymaterial, which is available to authorized users.

J. Lee � J. Kim � S. R. Min � H. Kim (&)

Plant Systems Engineering Research Center, Korea Research

Institute of Bioscience and Biotechnology (KRIBB),

125 Gwahangno, Yuseong-gu, Daejeon 305-806, Korea

e-mail: [email protected]

M. K. Kim

Division of Environment-Friendly Research,

Gyeongsangnam-do Agricultural Research and Extension

Service, Jinju 660-360, Korea

S. H. Hwang � S. S. Lim (&)

Department of Food Science and Nutrition, Hallym University,

1 Hallymdaehak-gil, Chuncheon 200-702, Gangwon-do, Korea

e-mail: [email protected]

J. M. Ahn

Breeding & Research Institute, Nongwoo Bio Co., LTD,

Yeoju, Kyonggi-do 469-885, Korea

S. U. Park

Department of Crop Science, College of Agriculture & Life

Sciences, Chungnam National University, 79 Daehangno,

Yuseong-gu, Daejeon 305-764, Korea

123

Mol Biol Rep (2014) 41:3487–3497

DOI 10.1007/s11033-014-3211-9

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CM Chorismatemutase

AS Anthranilate synthase

DAHPS 3-deoxy-D-arabino-heptulosonate 7-phosphate

synthase

CS Chorismate synthase

EPSP 5-enolpyruvylshikimate 3-phosphate synthase

AAT Alcohol acetyltransferase

Introduction

Melons (Cucumis melo) are one of the major horticultural

crops in the world, and include cantaloupe, honeydew, and

various mixed specialty types. It is a diploid species

(2n = 2X = 24) belonging to the Cucurbitaceae family,

which includes the cucumber, watermelon, and squash.

With its various phenotypes, the melon has been suggested

as a model for studying fruit ripening, sex determination,

and phloem physiology [1–4]. The oriental melon (C. melo

var. makuwa) called the ‘chamoe’ in Korean, has been used

traditionally for the treatment of various disorders as a liver

tonic, cardio-protective, anti-diabetic, anti-obesity, and

anti-cancer medicine [5–7]. The chamoe is a popular fruit

crop cultivated mainly in Asia and a high market value

crop in Korea. It is thought that melon and cucumber

originate in Asia. There are two major landrace of chamoe

in Korea: Sunghwan chamoe and Gotgam chamoe. The

Sunghwan chamoe has a green strain basis and a blue skin.

The Sunghwan chamoe contains more nutrients (such as

water, calcium, carbohydrates, and vitamins) than the

cultivar of chamoes. Moreover, it has greater disease

resistance compared to cultivar of chamoes [7]. With these

advantages, the whole genome of the melon was sequenced

using Sunghwan chamoe-derived double haploid lines [8].

Gotgam chamoe has green skin with distinctive green

stripes running from end to end, and a very thick, light

green flesh. The Gotgam chamoe has the aroma of a dried

persimmon, from which its name is derived. Breeders have

attempted to introduce the aroma of the Gotgam chamoe to

melons as well as chamoes because it adds more flavor and

savor. However, the constituents of this aroma as well as

the genotyping of this trait have not been studied in detail

up to now.

Aroma is an important trait of fresh and processed fruits

and vegetables. The aromas of fruits are determined by

distinctive combinations as well as the presence or absence

of volatile components [9–12]. Volatiles are naturally

produced by plants (flowers, fruits, vegetables, and herbs)

and animals. More than 300 compounds, including alco-

hols, aldehydes, esters, acids, ketones, and hydrocarbons

have been reported in fruits and vegetables [13–15]. The

volatile components of muskmelon have been identified

and include volatile aldehydes, alcohols, and an especially

large number of esters [16–18]. Volatile ester compounds

are key contributors to unique aromas, are produced during

ripening, and play an important role in determining the

final sensory quality of fruits [19, 20].

The shikimate pathway is considered as the common

route for the production of aromatic amino acids such as

phenylalanine, tyrosine, and tryptophan, which are found

only in microorganisms and plants, and never found in

animals [21, 22]. The shikimate pathway consists of seven

enzymes that catalyze the sequential conversion of ery-

throse 4-phosphate (E4P) and phosphoenolpyruvate (PEP)

to chorismate [23]. Chorismate, the end product of the

shikimate pathway, is the precursor of three aromatic

amino acids and several other aromatic compounds of

primary metabolism [24]. Chorismate is converted by

chorismatemutase (CM) and anthranilate synthase (AS) to

prephenate and anthranilate, respectively. The aromatic

amino acids are then biosynthesized from prephenate and

anthranilate. Phenylalanine is a general precursor of vari-

ous phenolic compounds [25]. Tryptophan is a precursor of

alkaloids, phytoalexins, indoleglucosinolates, and auxin.

Tyrosine is a precursor of isoquinoline alkaloids, pigment

betalains, and quinines [26, 27]. The key genes in the

shikimate and aromatic amino acid biosynthesis pathways

that have been identified in melons [28], apples [29],

grapes [30], oranges [31], pears [32], and strawberries [33]

enable us to investigate the expression levels of the genes

encoding the key enzymes for biosynthesis of aromatic

compounds.

In this study, we profiled and compared volatile com-

pounds in the different fruit parts of Gotgam (Korean

landrace) and Ohbokggul (cultivar) chamoes to elucidate

aroma of GgC, one of the breeding target traits. We also

analyzed the expression patterns of volatile biosynthesis

genes in both chamoes to investigate the correlations

between the metabolic pathway and transcription.

Materials and methods

Plant materials

Two chamoes (C. melo L. var. makuwa), Ohbokggul cha-

moe (Nongwoo Bio Co. F1 cultivar) and Gotgam chamoe

(C. melo var. makuwa, Nongwoo Bio Co. Accession No.

1638), were grown in a greenhouse at an experimental

farm, and obtained from Nongwoo Bio Co. (Korea) during

the fruiting season in October 2012. Ohbokggul and Got-

gam chamoes are differentiated by shape, size, color, and

flavor (Fig. 1). Three fruits of each chamoe were collected

and their peels, pulps, and stalks were separated. The

samples were frozen in liquid nitrogen, and stored at

-80 �C until analyzed.

3488 Mol Biol Rep (2014) 41:3487–3497

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Extraction of volatile compounds

A 1 cm long 50/30 lm polydimethylsiloxane/divinylbenzene/

carboxen-coated fiber was used for this analysis. The SPME

fiber was conditioned in a gas chromatography (GC) injection

port at 220 �C for 3 h and at 250 �C for 5 min prior to use.

The fresh fruit of each chamoe (2.0 g) were placed in a

15 mL thermo stated vial. During the SPME extraction pro-

cedure the SPME fiber was introduced for 30 min into the

thermostated vial (25 �C) with a rubber septum containing

2.0 g of the fresh fruit of each chamoe. The absorbed com-

ponent was injected into a GC by desorption at 220 �C for

1 min in the injector (splitless mode). The SPME procedure

was repeated three times and the results were presented as the

mean ± standard deviation.

Analysis of GC and GC-mass spectrometry (MS)

GC analysis was performed using an Agilent 6890 N GC

mainframe equipped with an HP-5 (30 m 9 0.32 mm ID,

film thickness 0.25 lm) fused-silica capillary column (Ag-

ilent, USA) and a flame ionization detector. The injector and

detector temperatures for each analysis were 220 and

280 �C, respectively. The carrier gas was nitrogen at a flow

rate of 1.0 ml/min. The column temperature was maintained

at 50 �C for 5 min and programmed as follows: rise from 50

to 85 �C at a rate of 2.5 �C/min, rise from 85 to 250 �C at a

rate of 5 �C/min, and hold at 250 �C for 5 min. GC–MS

analysis was performed on a GC/MSD Polaris Q (Thermo

Finnigan, USA) with an HP-5 (30 m 9 0.32 mm ID, film

thickness 0.25 lm) fused-silica capillary column (Agilent,

USA). Helium was used as the carrier gas at a flow rate of

1.0 ml/min. For GC–MS detection, an electron ionization

system with system energy 70 eV, trap current 250 lA, and

ion source temperature 200 �C was used. The oven tem-

perature program was the same as described for GC, and

injections were used in the splitless mode.

Identification of samples

The identification of the chemical constituents was based

on comparisons of their relative retention times. The

retention indices (RIs) were calculated for all compounds

using a homologous series of n-alkanes (C5-C20) under

same GC condition and by comparison of mass spectra

with NIST and WILEY library data of the GC–MS system

and with literature data. Total ion current chromatograms

were recorded in a mass range of 40–400 amu.

Analysis of reverse transcription-polymerase chain

reaction (RT-PCR)

Genbank accession numbers AB472682, AB472683,

AB472375, FJ604859, AB472685, AB472686, L47355,

AK226909, EU431334, NM_202520, M92353, U18968,

U18770, and NM_116495 were used as queries to identify

the homologous genes of the chamoe using our internal

transcriptome database of the chamoe (unpublished data).

To confirm the transcript level of selected genes, we per-

formed RT-PCR using primers listed in Table 1. For RT-

PCR analysis, total RNA was extracted with the RNeasy

Plant Mini Kit (QIAGEN, Germany), and cDNA was

synthesized using the iScript Select cDNA Synthesis Kit

(Bio-Rad, UK) according to the manufacturers’ instruc-

tions. The level of the C. melo actin gene (CmACT2) was

used as a loading control. RT-PCR reaction conditions

included 45 �C for 30 min, followed by 94 �C for 2 min;

28 cycles at 94 �C for 30 s; 52 �C for 30 s; 72 �C for

1 min; and a final extension step of 72 �C for 5 min. The

amplified gene products were sequenced and compared by

multiple alignments using ClustalW2 program (http://

www.ebi.ac.uk/Tools/msa/clustalw2/). The sequence vari-

ations were illustrated in Supplementary Fig. 1.

Analysis of quantitative real-time PCR

Quantitative real-time RT-PCR (qRT-PCR) was performed for

precise analysis of transcript levels. Primers that targeted each

gene and actin gene produced fragments of 80–90 bp were

designed using the Primer Quest computer program (http://eu.

idtdna.com/Scitools/Applications/Primerquest/). After cDNA

synthesis, qRT-PCR was performed using a SYBR� Green

Ohbokggul Chamoe Gotgam Chamoe

PEPU

S

Fig. 1 Photographs of Ohbokggul and Gotgam chamoes. PE peel,

PU pulp, S stalk

Mol Biol Rep (2014) 41:3487–3497 3489

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RT-PCR kit (IQ Sybr Green Super Mix) on a MiniOption

detection system (Bio-Rad, UK). Results were analyzed using

Bio-Rad software (GeneXpression Macro Chromo4) and the

comparative threshold cycle (Ct) method according to the

manufacturer’s instructions for data normalization.

Results and discussion

Profiling of volatile compounds in cultivar and landrace

chamoes

Two chamoes, Ohbokggul (cultivar; OC) and Gotgam

chamoe (Korean landrace; GgC), were harvested in Octo-

ber 2012, and their volatile compounds were profiled from

three parts: peels, pulps, and stalks (Fig. 1). A total of 62

volatile compounds were identified and quantified,

including 10 alcohols, 35 esters, 5 saturated hydrocarbons,

10 unsaturated hydrocarbons, and 2 ketones from both

chamoes. A total of 55 volatile compounds were identified

from the OC, and 43 volatile compounds from the GgC.

The most volatile compounds were detected in the stalk (46

compounds) of the OC and in the peel (32 compounds) of

the GgC. Ester compounds were the most different between

the two chamoes in terms of the number of detected vol-

atile compounds. Five saturated hydrocarbon compounds

were detected in common amongst both chamoes, but in

different amounts and patterns (Table 2).

Alcohols are known to contribute to the flavor and

aroma of fruit and serve as precursors for ester synthesis.

Table 1 Primers used in this study for RT-PCR and qRT-PCR analysis

Gene name Genbank

accession

number

Primer Sequence (50–30) Analysis

Forward primer Reverse primer

CmDAHPS AB472682 AACTTGCCCACCGAGTTGAT CATGAGATGTCCAGAAATCGG (RT-PCR)

TTGATGAGGCCCTTGGATTC ACTGCTCATAAGGCAAGAGTAAG (qRT-PCR)

CmDHQS AB472683 AGGCTATCGAGTCACGGCTT TGCCATAACAGTCGTGGGAA (RT-PCR)

AGGTGGTGTCATTGGTGATATG CCATAACAGTCGTGGGAATCTG (qRT-PCR)

CmDHD/SDH AB472375 GCCCACTGGCTTTCCAATAA TCAGACAGATAGGGCCTGACA (RT-PCR)

GCCCACTGGCTTTCCAATAA TCAGACAGATAGGGCCTGACA (qRT-PCR)

CmSK FJ604859 CGGAAAAACAACTGTGGGAA AACAAACTGGCGCATCAAAG (RT-PCR)

GTGCCGTAACGAGATCGATAAA GGACGAGAATTGGTTCCTACAG (qRT-PCR)

CmEPSPS AB472685 TTAACTGCCTTGCTGATGGC CTGGTGCCACAACCTTCAAC (RT-PCR)

GGTTGTGGCACCAGTAGTT CACTGTTCTCTGTCCAGGTTAC (qRT-PCR)

CmCS AB472686 GAGGTGGAGTTGGCTGTGTG GTCATATGTGGCATCGGCAT (RT-PCR)

CCCAATTTCGGAAGCTGATTTG CCGACATGTATCGGTCTCTTTC (qRT-PCR)

CmCM L47355 ATACAGTTGAAGCGGCATGG TGCGTAGGAAAAACCGAGTG (RT-PCR)

GCTATGGCGATGAGAGTAAGA CGAGCTTAGTGAGAGGGATAAG (qRT-PCR)

CmAS AK226909 CTTCCGGACACATTTTGTGG AAATCCATCAAGCTCCACCC (RT-PCR)

CTACGACAGCTTCACCTACAAC CCTCTTGGGTTCTTCCTCTTTAC (qRT-PCR)

CmPDH EU431334 ACACGGCCACGATCTAATCA TTTGGTGTTAATCGGCGAAG (RT-PCR)

CTCAATCGGAGCTCGTTTCTT GACTGATTCTGTGGAGAGGATTG (qRT-PCR)

CmAAT NM_202520 TAGTCGACATGAAGAGCGGC AAGTCCACGTTTTCAACCCC (RT-PCR)

GGTAATGCGGTAGTTGTTCCT CTTAGCCTTGGCCTTCCTAATC (qRT-PCR)

CmADT M92353 TAGCATGACGGATCTCTCGC TGTCGCGGAGGTTGTTAGAG (RT-PCR)

GGTTCCACAGCATCGTCATA CACCGGGAACACCTTGATAA (qRT-PCR)

CmPAI U18968 TTGTGGATAGTGCAACGGGT AGCCACCCATTTCTGCTTCT (RT-PCR)

GGGTGGGAGGTTCAATCATAA AGCTAAGAGCCACCCATTTC (qRT-PCR)

CmIGPS U18770 GGGAGGCTTTGAAAACTTGG CAGATTCGCCAACCACAATC (RT-PCR)

AGTATTTCCAGGGAGGCTTTG GCCTTTAGTCCGAGCATAGTAG (qRT-PCR)

CmTS NM_116495 GTGTGGCTCCGACATAATCG GCTCAATTTGATGCTTCCGA (RT-PCR)

GGTAGCGCGATGGTTAAGAT TGTGGGTATTCAAGAGACAAGAG (qRT-PCR)

CmACT2 AB033599 CCATTCTCCGTTTGGACCTT GCTCCGATGGTGATGACTTG (RT-PCR)

CTACGAACTTCCTGATGGACAAG CCAATGAGAGATGGCTGGAATAG (qRT-PCR)

3490 Mol Biol Rep (2014) 41:3487–3497

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0.0

7±

0.0

1P

ear

or

apri

cot

2-B

uth

yl-

4-e

thy

lb

enzo

ate

12

77

Nd

Nd

0.1

9±

0.0

2N

dN

dN

dF

ruit

y,

citr

us,

pea

ch

Eth

yl

no

nan

oat

e1

29

4N

d0

.13

±0

.01

0.2

9±

0.0

3N

dN

dN

dF

ruit

yro

sew

axy

rum

win

e

Mol Biol Rep (2014) 41:3487–3497 3491

123

Page 6

Ta

ble

2co

nti

nu

ed

Vo

lati

leco

mp

ou

nd

RI

Pea

kar

ea(%

)O

do

rd

escr

ipti

on

Oh

bo

kg

gu

lC

ham

oe

Go

tgam

Ch

amo

e

PE

PU

SP

EP

US

tra

ns-

2-H

exen

yl

acet

ate

13

08

0.1

9±

0.0

20

.22

±0

.02

0.3

1±

0.0

30

.03

±0

.01

0.0

5±

0.0

10

.06

±0

.01

Wax

y,

ban

ana-

lik

e

Oct

-1-e

n-3

-yl

acet

ate

14

78

0.1

1±

0.0

10

.58

±0

.06

Nd

Nd

Nd

Nd

Gre

en,

eart

hy

3-A

cety

lox

yb

uta

n-2

-yl

acet

ate

14

95

Nd

Nd

0.5

7±

0.0

6N

dN

dN

dS

wee

tet

her

-lik

e

Die

thy

l-2

-all

yl-

2-h

yd

rox

y-

pen

tan

edio

ate

15

52

Nd

Nd

0.2

4±

0.0

3N

dN

dN

dN

on

ere

po

rted

Eth

yl

do

dec

ano

ate

15

97

0.1

9±

0.0

20

.03

±0

.01

Nd

Nd

Nd

Nd

Pea

nu

t-li

ke

Lin

alo

ol

pro

pan

oat

e1

62

40

.06

±0

.01

0.0

1±

0.0

10

.16

±0

.02

Nd

Nd

Nd

Fre

shly

fru

ity

,fl

ora

l

a-T

erp

iny

lp

rop

ano

ate

17

03

0.0

8±

0.0

10

.07

±0

.01

0.3

±0

.03

0.0

4±

0.0

1N

d0

.52

±0

.05

Pin

ewo

od

y,

flo

ral

Iso

bo

rny

lp

rop

ano

ate

17

63

Nd

Nd

0.2

6±

0.0

30

.07

±0

.01

Nd

0.0

5±

0.0

1P

un

gen

tp

ine

cam

ph

or

wo

od

yla

ven

der

Ner

yl

pro

pan

oat

e1

79

4N

dN

d0

.23

±0

.02

0.0

4±

0.0

1N

d0

.04

±0

.01

Fru

ity

,ja

mm

y

Ger

any

lp

rop

ano

ate

18

34

0.0

±0

.01

Nd

0.1

5±

0.0

20

.05

±0

.01

0.0

3±

0.0

10

.14

±0

.01

Ro

se,

lem

on

aro

ma

[(Z

)-7

-met

hy

ltet

rad

ec-8

-en

yl]

acet

ate

18

92

Nd

Nd

0.1

5±

0.3

1N

dN

dN

dC

anta

lou

pe-

lik

e,fr

uit

y

Eth

yl

hex

adec

ano

ate

19

26

Nd

3.2

8±

0.3

1N

dN

dN

dN

dS

oft

,w

axy

Des

yl

ben

zoat

e2

03

0n

d0

.02

±0

.01

0.0

9±

0.0

10

.14

±0

.01

nd

0.1

6±

0.0

2N

on

ere

po

rted

Eth

yl

oct

adec

ano

ate

21

96

0.1

±0

.01

0.4

4±

0.0

50

.07

±0

.01

0.2

±0

.02

0.0

6±

0.0

10

.06

±0

.01

Wax

y

Eth

yl

iso

allo

cho

late

23

04

0.2

5±

0.0

3N

d0

.04

±0

.01

Nd

Nd

Nd

Pen

etra

tin

g,

Sic

ken

ing

Su

bto

tal

80

.21

–5

.04

76

.26

–6

.32

77

.24

–7

.45

78

.3–

8.8

67

3.7

6–

8.1

67

9.7

1–

7.8

1

Sat

ura

ted

hy

dro

carb

on

7-M

eth

yl

hep

tad

ecan

e1

28

8N

dN

d0

.21

±0

.02

0.0

8±

0.0

1N

d0

.06

±0

.01

No

ne

rep

ort

ed

n-N

on

adec

ane

17

93

0.0

7±

0.0

1N

d0

.26

±0

.03

0.0

5±

0.0

1N

d0

.04

±0

.01

No

od

or

6-M

eth

yl

oct

adec

ane

18

63

0.0

5±

0.0

1N

d0

.18

±0

.02

0.0

5±

0.0

1N

d0

.04

±0

.01

Eth

er

n-D

oco

san

e2

20

00

.05

±0

.01

0.0

2±

0.0

10

.36

±0

.04

0.0

3±

0.0

10

.07

±0

.01

0.0

7±

0.0

1N

on

ere

po

rted

4-M

eth

yl

do

cosa

ne

22

20

0.0

2±

0.0

10

.02

±0

.01

0.2

1±

0.0

10

.05

±0

.01

Nd

Nd

Wax

yty

pe

Su

bto

tal

0.1

9–

0.0

40

.04

–0

.02

1.2

2–

0.1

20

.26

–0

.05

0.0

7–

0.0

10

.21

–0

.04

Un

satu

rate

dh

yd

roca

rbo

n

Hep

ta-1

,2,4

,6-t

etra

ene

65

5N

dN

dn

dN

dN

d0

.85

±0

.08

Un

ple

asan

t

5-M

eth

yl-

3-o

cten

e8

38

0.2

6±

0.0

20

.48

±0

.05

nd

Nd

Nd

Nd

No

od

or

2-M

eth

yl-

2-d

ecen

e1

00

0N

dN

dn

dN

dN

d0

.32

±0

.03

Un

ple

asan

t

1,3

,5-H

epta

trie

ne

10

05

Nd

Nd

Nd

Nd

5.0

1±

0.8

0N

dS

wee

t,b

anan

a,fr

uit

y

1,3

,7-O

ctat

rien

e-5

-yn

e1

00

8N

d0

.3±

0.0

30

.07

±0

.01

Nd

Nd

Nd

Un

ple

asan

t

3-P

enty

l-1

-cy

clo

hex

ene

11

50

0.2

1±

0.0

2N

dN

dN

dN

dN

dF

lora

l

1,3

,5,7

-Cy

clo

oct

atet

raen

e1

19

60

.16

±0

.02

0.1

6±

0.0

20

.16

±0

.02

0.1

4±

0.0

10

.11

±0

.01

0.1

7±

0.0

2S

wee

to

lefi

n,

aro

ma

tra

ns-

Oci

men

e1

24

03

.04

±0

.81

5.9

2±

0.3

14

.79

±0

.70

4.1

8±

0.4

14

.29

±0

.71

Nd

Fre

sho

ran

ge

juic

e

3492 Mol Biol Rep (2014) 41:3487–3497

123

Page 7

Consequently, their composition reflects the presence of

esters in the fruit [34]. Most of the alcohol compounds

were identified in the stalk part of the OC, and were 12.03

times higher there than in the stalk part of the GgC. Two

times more alcohol compounds were detected in peel part

of the GgC compared to that of the OC. The most abundant

alcohol compound detected in the peel, pulp, and stalk of

the OC was 3-methyl-1-butanol, but it was not detected in

stalk of the GgC. Among the detected alcohol compounds,

3,3,6-trimethyl hepta-1,5-dien-4-ol and 2,4,6-triisopropyl-

phenol were found only in the OC; however, eugenol was

detected only in the GgC (Table 2).

Esters are known to impart a fruity characteristic or

sweet odor in apples, strawberries, and pears [35, 36]. Our

results suggested that esters were the predominant volatile

compounds in chamoes. The number of ester compounds

was higher in the OC than the GgC, but total amounts of

esters were similar. The highest amount of esters was

detected from the peel part of the OC. The most dominant

ester in both chamoes was 2-methyl-1-butyl acetate, with a

peak range of 36.57–73.43 %. This compound was detec-

ted in different amounts depending on the part of the fruit

tested; however, it seemed to contribute to the majority of

the fruity odor of both chamoes. A total of 12 volatile

esters were identified only in the OC, and the total amount

of these specific compounds was higher in the pulp and

stalk parts which contribute fruity, citrus, and soft odors.

The GgC showed three specific ester compounds that were

detected mainly in the stalk, and these compounds provide

sweet fruity, winy, and pineapple odors (Table 2). Benzyl

acetate, which has been reported as the most important

synthetic aroma chemical in melon [37] and contributes

sweet and fruity flavors, was detected only in the GgC in

this study.

Beaulieu [29, 38] reported that methyl, ethyl, butyl, hexyl,

nonenyl, and benzyl acetates; butanoates; methylpropa-

notes; and hexanoates are the dominant esters in melon. In

apples, dominant esters are ethyl, butyl and hexyl acetates;

butanoates; and hexanoates [39]. Strawberries are comprised

primarily of methyl and ethyl acetates; butanoates; and

hexanoates [40, 41]. In our study, esters were dominated by

2-methyl-1-butyl, n-buthyl, and 3-methylbutyl acetate;

ethyl-2-methyl butanoate; and methyl formate in both cul-

tivar and landrace chamoes. Ester compounds such as

3-methylbutyl propanoate, ethyl hexadecanoate, 3-meth-

ylbutyl butanoate, and pentyl acetate were found only in

Ohbokggul chamoe, whereas ethyl butyrate and n-amyl

acetate were specific to Gotgam chamoes (Table 2). Esters

were the predominant compounds of our chamoes, but ester

compounds were different in each chamoe type, thus

reflecting their fruity flavor characters.

As shown in Table 2, all five saturated hydrocarbons

detected were identified mostly in the stalk of theTa

ble

2co

nti

nu

ed

Vo

lati

leco

mp

ou

nd

RI

Pea

kar

ea(%

)O

do

rd

escr

ipti

on

Oh

bo

kg

gu

lC

ham

oe

Go

tgam

Ch

amo

e

PE

PU

SP

EP

US

Bic

ycl

o[4

.4.0

]dec

a-1

,3,5

,7,9

-pen

ten

e1

24

20

.12

±0

.01

0.1

8±

0.0

2N

dN

dN

dN

dP

un

gen

t

1,(

E)-

8,(

Z)-

10

-Hex

adec

atri

ene

18

50

0.1

8±

0.0

2N

dN

dN

dN

dN

dU

np

leas

ant

Su

bto

tal

3.9

7–

0.9

7.0

4–

0.4

35

.02

–0

.73

4.3

2–

0.4

29

.41

–1

.52

1.3

4–

0.0

5

Ket

on

e

1-P

hen

yl

pro

pan

-2-o

ne

11

54

5.4

8±

0.8

17

.16

±0

.62

6.1

5±

0.4

27

.13

±0

.90

7.5

6±

0.7

62

.96

±0

.30

Ro

tten

egg

smel

l

4-H

yd

rox

yb

uta

no

icac

idla

cto

ne

16

18

0.5

4±

0.0

50

.8±

0.0

80

.33

±0

.02

2.0

5±

0.2

51

.45

±0

.35

3.4

9±

0.5

9C

ream

y

6.0

2–

0.8

67

.96

–0

.70

6.4

8–

0.4

49

.18

–1

.15

9.0

1–

1.1

16

.45

–0

.89

To

tal

94

.51

–7

.26

94

.38

–7

.70

96

.10

–9

.69

5.1

7–

10

.84

94

.83

–1

1.0

48

8.2

2–

8.8

6

Bo

ldv

alu

esin

dic

ate

tota

lam

ou

nt

of

vo

lati

leco

mp

ou

nd

s

RI

rete

nti

on

ind

ex,

PE

pee

l,P

Up

ulp

,S

stal

k,

Nd

no

td

etec

ted

Mol Biol Rep (2014) 41:3487–3497 3493

123

Page 8

Ohbokggul chamoe (most abundant) and the peel of the

Gotgam chamoe (less abundant). The detected high

molecular weight compounds, 4-methyl docosane and

6-methyl octadecane, have been reported to donate waxy

and acetone-like odors. Five out of ten unsaturated hydro-

carbon compounds were detected only in the Ohbokggul

chamoe with a peak range of 0.07–0.48 %, and five com-

pounds were specific to the Gotgam chamoe with a peak

range of 0.32–5.01 %. 1,3,5-heptatriene, which gives sweet

and fruity odors was detected only in the pulp of the Gotgam

chamoe. Trans-ocimene, which provides a fresh orange

juice odor, was abundantly detected in both chamoes, but

not in the stalk of the Gotgam chamoe. Two ketones were

identified and the content of 4-hydroxy butanoic acid lac-

tone (which gives a creamy flavor) was higher in the Gotgam

chamoe than the Ohbokggul chamoe (Table 2). As a result,

volatile compounds were differently present within the

different parts of the two chamoes, and this contributed to

the aromatic differences between cultivar and native melon.

These data demonstrate the kinds and quantities of aroma

compounds present within different parts, and that the

aroma of chamoe was not related to any single compound,

but due to the combinations of volatiles.

Expression analysis of shikimate pathway genes

The transcript levels of shikimate pathway genes were

compared in Ohbokggul and Gotgam chamoe by RT-PCR

and qRT-PCR. The six major genes in the pathway iden-

tified within the chamoe were CmDAHPS, CmDHQS,

CmDHD/SDH, CmSK, CmEPSPS, and CmCS. The first

enzyme involved in the shikimate pathway is 3-deoxy-D-

arabino-heptulosonate 7-phosphate synthase (DAHPS)

(Fig. 2a). DAPHS is regulated through allosteric feedback

inhibition by the final products of the shikimate pathway

(tyrosine, tryptophan, and phenylalanine) in microbes [42,

43], whereas DAHPS enzymes are regulated by redox

regulation [44] in plants. This protein condenses from PEP

and E4P to produce DAHP. As shown in Fig. 2b,

CmDAHPS was expressed at a moderate level in the three

parts of the Ohbokggul chamoe fruit, but the expression

was decreased gradually from peel to stalk of the Gotgam

chamoe. CmDHQS and CmEPSPS were highly expressed

in the peel and pulp of the Gotgam chamoe, two times and

three times, respectively, whereas transcript levels were

similar in stalk part (Fig. 2b). The expression level of

CmDHD/SDH and CmSK were low compared to the

A

Phosphoenolpyruvate

3-deoxy-D-arabinoheptulosonate 7-phosphate

DHQS

3-dehydroquinate

DHD/SDH

3-dehydroshikinate

Shikimate

Shikimate 3-phosphate

SK

5-enolpyruvylshikimate 3-phosphate

Chorismate

EPSPS

CS

Phosphoenolpyruvate Erythrose 4-phosphate

BOC

CmDHD/SDH

CmACT2

GgC

PE PU S

OCCmDHQS

CmACT2

GgC

PE PU S

OCCmSK

CmACT2

GgC

PE PU S

OCCmEPSPS

CmACT2

GgC

PE PU S

OCCmCS

CmACT2

GgC

PE PU S

OCCmDAHPS

CmACT2

GgC

PE PU S

Rel

ativ

e m

RN

A le

vel

OC GgC Rel

ativ

e m

RN

A le

vel

OC GgC Rel

ativ

e m

RN

A le

vel

OC GgC

Rel

ativ

e m

RN

A le

vel

OC GgC Rel

ativ

e m

RN

A le

vel

OC GgC

Rel

ativ

e m

RN

A le

vel

OC GgC

Fig. 2 Shikimate pathway and expression of CmDAHPS, CmDHQS,

CmDHD/SDH, CmSK, CmEPSPS, and CmCS in different parts of the

chamoe. a The Shikimate pathway converts phosphoenolpyruvate and

erythose 4-phosphate into chorismate in higher plants [45]. Enzyme

names are given on the right side of the arrows. b Total RNA was

isolated from the peel, pulp, and stalk (individually) and analyzed by

RT-PCR and qRT-PCR using gene specific primers. CmACT2 mRNA

was amplified to confirm that a constant amount of total mRNA was

used and ubiquitin was equally expressed in all samples. Each value is

the mean of three replicates, and error bars indicate standard

deviations. PE peel, PU pulp, S stalk, OC Ohbokggul chamoe, GgC

Gotgam chamoe

3494 Mol Biol Rep (2014) 41:3487–3497

123

Page 9

A Shikimate pathway

Chorismate

Prephenate Anthranilate

ASCM

Arogenatep-hydroxyphenylpyruvate

PDH

Tyrosine

AATADT

Phenylalanine

Phosphoribosylanthranilate

IGPS

L-(o-carboxyphenylamino)-l-deoxyribulose-5-phosphate

Indole-3-glycerol phosphate

Indole

TS

TS

PAI

Tryptophan

BOC

GgC

PE PU S

CmAAT

CmACT2

CmPDH

CmACT2

OC

GgC

PE PU S

OC

CmCM

CmACT2

GgC

PE PU S

OC

GgC

PE PU S

CmAS

CmACT2

OC

GgC

PE PU S

CmPAI

CmACT2

OC

GgC

PE PU S

CmIGPS

CmACT2

OC

GgC

PE PU S

CmTS

CmACT2

OC

GgC

PE PU S

CmADT

CmACT2

Rel

ativ

e m

RN

A le

vel

OC GgC Rel

ativ

e m

RN

A le

vel

OC GgC Rel

ativ

e m

RN

A le

vel

OC GgC

Rel

ativ

e m

RN

A le

vel

OC GgC

Rel

ativ

e m

RN

A le

vel

OC GgC

Rel

ativ

e m

RN

A le

vel

OC GgC

Rel

ativ

e m

RN

A le

vel

OC GgC Rel

ativ

e m

RN

A le

vel

OC GgC

Fig. 3 Biosynthesis pathways

of the aromatic amino acids in

plants and expression of CmCM,

CmPDH, CmAAT, CmADT,

CmAS, CmPAI, CmIGPS, and

CmTS in different parts of the

chamoe. a Schematic

representation biosynthesis

pathways of the aromatic amino

acids in plants [45]. b Total

RNA was isolated from the peel,

pulp, and stalk (individually)

and analyzed by RT-PCR using

gene specific primers. CmACT2

mRNA was amplified to

confirm that a constant amount

of total mRNA was used and

ubiquitin was equally expressed

in all amples. Each value is the

mean of three replicates, and

error bars indicate standard

deviations. PE peel, PU pulp,

S stalk, OC Ohbokggul chamoe,

GgC Gotgam chamoe

Mol Biol Rep (2014) 41:3487–3497 3495

123

Page 10

expression of other genes investigated. Those genes were

rarely expressed in the pulp and stalk, and shared a similar

expression pattern between Ohbokggul and Gotgam

chamoes. The last step of the shikimate pathway is cata-

lyzed by chorismate synthase (CS), which converts 5-e-

nolpyruvylshikimate 3-phosphate synthase (EPSP) to

chorismate (Fig. 2a). CmCS was the most expressed gene

among the six genes, and showed higher transcript levels in

the stalk of the Gotgam chamoe compared to that of the

Ohbokggul chamoe, while the other two parts showed

similar expression levels in both chamoes (Fig. 2b). Con-

sidering the expression patterns of CmEPSPS and CmCS,

some chorismate can accumulate in higher levels in the

pulp and stalk of the Gotgam chamoe than in those of the

Ohbokggul chamoe.

Differential expression of aromatic amino acid-related

genes

To compare the aroma-related gene expression in the both

chamoes, transcripts of eight aromatic amino acid biosyn-

thesis genes (Fig. 3a) [45] were identified from the chamoe

genome and monitored (Fig. 3). Identical expression pat-

terns were detected within the fruit parts for CmCM,

CmPDH, CmAAT, and CmADT from both chamoes. Their

expression was higher in all parts of the fruit compared to

the expression of the remaining four tryptophan biosyn-

thesis genes (Fig. 3b). The expression of CmAAT in the

pulp of the both chamoes was the highest among all of the

aromatic amino acid biosynthesis genes (Fig. 3b). Alcohol

acetyltransferase (AAT) is known to be a key enzyme in

aroma biochemistry since it catalyzes the last step in the

biosynthesis of volatile esters [46]. As previously described

by Lucchetta (2007) AAT gene is positively regulated by

ethylene during ripening of the melon [47].

Regulated enzyme CmADT was expressed higher in all

three parts of the Gotgam chamoe (Fig. 3b) than in the

Ohbokggul chamoe. This result suggests that the phenyl-

alanine accumulation level is higher in the Gotgam chamoe

than the Ohbokggul chamoe. In general, expression levels

of the tryptophan biosynthesis genes were lower in the both

chamoes than the expression levels of tyrosine and phen-

ylalanine biosynthesis genes. Those genes showed low

expression patterns in the stalk and higher expression

patterns the peel of the both chamoes. The first committed

step of tryptophan biosynthesis is generation of anthrani-

late from chorismate, catalyzed by AS (Fig. 3a). The

transcript level of CmAS was reduced from peel to stalk in

both chamoes. CmPAI expression was similar in peel and

stalk but reduced in the pulp of both chamoes. Expression

patterns of CmIGPS and CmPAI within the fruit parts were

different in the two chamoes. CmIGPS was expressed

higher in the peel and stalk versus the pulp of the

Ohbokggul chamoe while the expression was gradually

reduced from peel to stalk in the Gotgam chamoe. CmTS

exhibited a distinct Gotgam chamoe-specific expression

pattern, especially in the peel and pulp (Fig. 3b). These

collective results demonstrate that phenylalanine and

tyrosine present more in edible parts of the chamoes, and

that phenylalanine and tryptophan are synthesized more in

the Gotgam chamoe (Korean landrace) than the Ohbokggul

chamoe (cultivar). We suggest that in general the accu-

mulated amount of tryptophan may be low in chamoes.

This transcriptional difference in the aromatic amino acid

biosynthesis genes may be the cause of the variation in fruit

aromas among the chamoe varieties.

Acknowledgments This work was financially supported by Grants

from the Next-Generation Bio Green 21 Program (No. PJ008200),

Cabbage Genomics assisted breeding supporting Center (CGC)

research programs, and Hallym University Research Fund (HRF-

201302-006) funded by Rural Development Administration and

Ministry for Food, Agriculture, Forestry and Fisheries of the Korean

Government, and Hallym University, respectively.

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