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Profiling of Terpene Metabolome in Carrot Fruits of Wild (Daucus carota L. ssp. carota) Accessions and Characterization of a Geraniol Synthase Mosaab Yahyaa, Muhammad Ibdah, Sally Marzouk, and Mwafaq Ibdah J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03596 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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Journal of Agricultural and Food Chemistry

Profiling of Terpene Metabolome in Carrot Fruits of Wild (Daucus carota L. ssp. carota) Accessions and Characterization of a Geraniol Synthase

Mosaab Yahyaa1, Muhammad Ibdah2, Sally Marzouk1, Mwafaq Ibdah1* 1

NeweYaar Research Center, Agriculture Research Organization, P.O.Box 1021, Ramat

Yishay, 30095, Israel. 2

Sakhnin College Academic College for Teacher Education, Israel.

*Corresponding Organization,

author: P.O.

Box

Newe

Yaar

1021,

Research

Ramat

Center,

Yishay,

Agricultural

30095,

ISRAEL;

Research E-mail:

[email protected]; Telephone: 00972-4-953-9509; Fax:00972-4-953-9509

1 ACS Paragon Plus Environment


1

ABSTRACT

2

Fruits from wild carrot (Daucus carota L. ssp. carota) have been used for medicinal

3

purposes since ancient times. The oil of its seeds, with their abundant monoterpenes and

4

sesquiterpenes, has drawn attention in recent years because of their potential

5

pharmaceutical application. A combined chemical, biochemical and molecular study was

6

conducted to evaluate the differential accumulation of terpene volatiles in carrot fruits of

7

wild accessions. In this work, we report on a similarity-based cloning strategy

8

identification and functional characterization of one carrot monoterpene terpene

9

synthases, WtDcTPS1. Recombinant WtDcTPS1 protein produces mainly geraniol, the

10

predominant monoterpene in carrot seeds of wild accession 23727. Our results suggest a

11

role for WtDcTPS1 gene in biosynthesis of carrot fruits aroma and flavor compounds.

12 13 14 15 16 17 18 19 20 21

KEYWORDS: Daucus carota L. ssp. carota, wild carrot fruits, auto-HS-SPME-GC-MS,

22

volatile terpenes, RACE, geraniol synthase.

23


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INTRODUCTION

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The genus Daucus is a member of the Apiaceae (Umbelliferae) family, which is

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one of the most important families of angiosperms from an economical point of view.1

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This family comprises such important crop species as cultivated carrot (D. carota subsp.

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sativus Hoffman), parsley (Petroselinum crispum), celery, fennel (Foeniculum vulgare),

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cumin (Cuminum cyminum L.) and many others. 1 Despite the agricultural and economic

30

importance of D. carota, only recently the complete D. carota genome was published. 2

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Wild carrot (Daucus carota L. ssp. carota), also known as Queen Anne’s lace,

32

originated in Central Asia and spread in early times to the Mediterranean, Australia,

33

Americas, and New Zealand.3 Wild carrot is the ancestor of the cultivated carrot (Daucus

34

carota ssp. sativus). Wild and cultivated carrots intercross freely, which has significant

35

implications both for historical development of the modern carrot and for the future of

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carrot.1, 4 It is well known that both wild and cultivated carrots are predominantly cross-

37

pollinated by a large diversity of insects.5,

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cultivated and wild carrot may be very high when both spatial distribution and flowering

39

overlap.7, 8

6

Therefore, gene flow frequency between

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Wild carrot is the most widespread species of the genus Daucus. 1 The D. carota

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species shows great morphological plasticity, resulting in the presence of a range of

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distinct phenotypes.1 Wild carrots have small spindle-shaped, white, slim roots that are

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aromatic and harsh with an unpleasant taste. In some countries, it is considered a weed.7, 9

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The fruits of the wild carrot are aromatic and have been used since ancient times as

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medicine for the treatment of a wide range of diseases.10 Moreover, the essential oils of

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D. carota were found to exhibit antibacterial activity, against Bacillus subtilis,



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Staphylococcus aureus, Escherichia coli, and Campylobacter jejuni.11, 12 In foods, wild

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carrot oil is used to flavor alcoholic and non-alcoholic beverages. In manufacturing,

49

essential oil is used as a fragrance in soaps, detergents, creams, lotions, and perfumes.13

50

Apiaceaeous plants are rich in volatile, which are constitutes essential oils present in

51

leaves, fruits, and seeds. In the most studied species, monoterpenes dominated of all

52

volatile components. They are usually followed by sesquiterpenes.1, 11, 14 The qualitative

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and quantitative terpene composition of D. carota seeds is mostly dependent on the

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geographical region,15, 16 the stage of development,17 and the part of the plant.14, 18-20

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Terpenes constitute a large class of secondary metabolites and have a wide variety

56

of functions, e.g. roles in direct defense against herbivores,21,

22

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defense compounds by attracting predators or parasitoids of phytophagous pests.23

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Terpenes represent major components of floral scents and essential oils of herbs,24 and

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are important in determining the quality and nutraceutical properties of horticultural food

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products, including the taste and aroma of wine, melon, and carrot.25-27 Terpenes are

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produced by action of terpene synthases (TPSs), which accept geranyl pyrophosphate

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(GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) as

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substrates and convert them into different monoterpene, sesquiterpene, and diterpene

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skeletons, respectively.28, 29

as well as in indirect

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Despite the pharmaceutical and economic importance of the chemical

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composition of essential oils of the Daucus species,11, 14, 15, 30 little is known about the

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biosynthesis of its major constituents in wild carrot fruits. Thus, we have begun an

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investigation of the volatile chemical compositions of four different species of wild carrot

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fruits by means of auto-headspace solid phase micro extraction-gas chromatography-


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mass spectrometry. Also, we investigated the enzymes responsible for terpene

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biosynthesis in these fruits. Here, we report the identification and characterization of one

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wild carrot terpene synthase gene, i.e. wild type Daucus carota terpene synthase 1

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(WtDcTPS1) which encodes an enzyme catalyzing the formation of the monoterpene

74

alcohol geraniol. We show that WtDcTPS1 is expressed in all different tissues

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investigated in this study and is associated with the production of high amounts of

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geraniol in carrot fruits of wild accession 23727.

77 78

MATERIALS AND METHODS

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Chemicals

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Unlabeled FPP and GPP (1 mg/mL), terpene standards, other chemicals, and

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reagents were purchased from Sigma-Aldrich unless noted otherwise.

82

Plant Species

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We examined nine accessions of four Daucus species (D. carota L.; D. broteri

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Ten.; D. glaber (Forssk.) Thell.; D. aureus Desf.) obtained from “Israel Plant Gene

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Bank”. Seeds were previously collected from different area of Israel (Table 1). Wild

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carrot plants were grown at the Newe Yaar Research Center, Agricultural Research

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Organization, in northern Israel, under standard field irrigation and fertigation conditions.

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Freshly harvested tissues (roots, leaves, flowers, fruit pericarp, and whole intact fruits)

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were crushed in liquid nitrogen and stored at -80 °C for volatile analysis.

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Since, carrot fruits are commonly named seeds, and we did not excised seeds

91

from fruits but we used whole intact fruits for analyses, we will use the term fruits for

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whole intact fruits from here on.



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Extraction of Volatile Compounds from Carrot Fruits of Wild Accessions

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Five biological replicates of carrot fruits (0.5 g) from plants of different

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accessions were ground into a uniform powder under liquid nitrogen with a mortar and

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pestle. The powders were placed in a 20 mL SPME vial containing 1 g NaCl, 7 mL 20%

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(w/v) NaCl and 10 ppm of 2-heptanone as an internal standard, sealed and placed in 4 ºC

98

until used. Headspace sampling was conducted utilizing a 65 µm fused silica fiber coated

99

with polydimethylsiloxane/divinylbenzen (PDMS/DVB) (Supleco).27, 31 After 40 min at

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65 °C, and. under gentle stirring, the SPME syringe was introduced into the injector port

101

of the GC-MS apparatus for further analysis.

102 103

Auto-Head Space (HS)-SPME-GC-MS Analysis of Carrot Fruits of Wild Accessions

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Volatile Compounds

105

Volatile compounds were analyzed on a GC-MS apparatus (Agilent Technologies

106

CA, USA) equipped with an Rtx-5SIL MS (30 m x 0.25 mm x 0.25 µm) fused-silica

107

capillary column. He (1 mL min-1) was used as a carrier gas. The injector temperature

108

was 250 °C, set for splitless injection. The oven was set to 50 °C for 1min, and then the

109

temperature was increased to 220 °C at a rate of 5 °C min-1. The detector temperature was

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280 °C. The mass range was recorded from 41 to 450 m/z, with electron energy of 70eV.

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Identiﬁcation of the main components was done by comparison of mass spectra and

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retention times with those of authentic standards and supplemented with a Wiley GC-MS

113

library.

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For the construction of the calibration curves, a mixture of straight-chain alkanes

115

(C7-C23) was run under the above mentioned conditions to determine retention indices.


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The amount of each compound in the sample was calculated as (peak area x internal

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standard response factor) divided by (response factor x internal standard peak area).27, 31

118 119

Cell-Free Extracts Derived from Carrot Fruits of Wild Accession

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The mono-, and sesquiterpene synthase activity was extracted from 2 g of wild

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carrot fruits accession 23727 that were grown under normal conditions, ground in liquid

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nitrogen with extraction buffer, and stored at -80 °C. All extraction and purification steps

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were performed at 4 °C. Extraction buffer contained 50 mM Bis-Tris, pH 6.9, 10% (v/v)

124

glycerol, 10 mM dithiothretiol (DTT), and 5 mM sodium pyrosulfite (Na2S2O5). The

125

crude protein extract was passed through two layers of Micracloth (Calbiochem) and

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centrifuged for 20 min at 12,000g at 4 °C. The pellet was discarded, the supernatant was

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passed over DE52 beads (pre-equilibrated with 50 mM Bis-Tris, pH 6.9) to remove

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residual impurities, and proteins were eluted with a stepwise NaCl gradient (100, 250,

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and 500 mM NaCl in 50 mM Bis-Tris, pH 6.9) with monitoring of mono-, and

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sesquiterpene synthase enzyme activity.32

131 132 133

Isolation and Characterization of Wild Carrot Fruits Terpene Synthase Alignment of several previously characterized angiosperm terpene synthases, 1, 2, 27

134

including Daucus carota DcTPSs

enabled degenerated primer design based on

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several highly conserved regions, as described by Chang et al.33 Forward primer

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WtDcTPS-d-F (5´-CARAGRTTRGGNG TNGCNTAYCAYTTY-3´), and reverse primer

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WtDcTPS-d-R (5’-YTTRAARTANACNCCNAGDATCCA-3’) were based on the

138

QRLGV(A/S)Y(Q/H)F and WILGVYFE motifs, respectively.



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RNA from wild carrot of fruits with accession number 23727 was isolated using

140

the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich). For producing a cDNA clone, 5

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µg of total RNA from wild mature fruits was reversed transcribed with RevertAid First

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Strand cDNA Synthesis Kit (Thermo Fisher scientific). The PCR product was run on an

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agarose gel. A band of an 300 base pair (bp) was gel purified, cloned into pJET 1.2/blunt

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cloning vector (Thermo Fisher scientific) and sequenced.

145 146

RACE Strategies

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Rapid amplification of 5’- and 3’- cDNA ends (RACE) was performed to attain

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WtDcTPS cDNA using a SMARTer RACE 5’/3’ Kit (Clontech Laboratories, Inc. USA)

149

according to manufacturer’s instructions. For the 5’-RACE, PCR was performed with a

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gene-specific primer, (5’-RACE: 5’-CCCGGCTTGCCTTAGTAGCCGGAAACA-3’).

151

For the 3’-RACE, PCR was performed with a gene-specific primer, 3’-RACE: 5’-TGG

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TGGAGTGTTACTTCTGGATCCTGGGCG-3’) RACE PCR products were analyzed on

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agarose gels and purified using an Agarose Gel DNA Extraction Kit, cloned into pJET

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1.2/blunt cloning vector (Thermo Fisher scientific) and sequenced.

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The full-length of WtDcTPS1 cDNA sequence was submitted to the NCBI blastp

156

to perform homologous sequence search. Multiple sequence alignment was analyzed with

157

clustalx software. A phylogenetic tree was constructed with the neighbor-joining method

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in Molecular Evolution Genetics Analysis (MEGA 6) software version 6.34 The amino

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acid sequences used in other species were acquired from the NCBI GenBank database

160

(http://www.ncbi.nlm.nih.gov/protein).

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Cloning and Expression of a cDNA of the WtDcTPS1

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Based on sequence information, two gene-specific primers were designed

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corresponding to the 5′-end (5′-ATGGCCCTCCCAGCTCTGTTTT-3′) and 3′-end (5′-

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CTGGCTAAGAGTAAAGGGTTCGACC-3′) of the wild carrot fruits WtDcTPS1

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nucleotide sequence. RNA from wild carrot fruits (accession 23727) was isolated using

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the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich). For cloning, 5 µg of total RNA

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was reversed transcribed with RevertAid First Strand cDNA Synthesis Kit (Thermo

169

Fisher scientific) and the corresponding cDNA was amplified to yield a ~1779-bp

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fragment. The cDNA was ligated into the vector pEX5-CT⁄TOPO TA (Invitrogen,

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Carlsbad, CA, USA) to create a fusion of the open-reading frame with a His tag-coding

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extension at the C-terminus. The plasmid was transferred into Escherichia coli BL21

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(DE3) cells. Protein expression was induced with 1 mM isopropyl-1-thio-β-D

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galactopyranoside at 18 °C for 14 h. Bacteria were lysed and soluble protein purified by

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nickel-chromatography and assayed for purity by SDS-PAGE as previously described.27

176 177

Assay for Terpene Synthase Activity Enzyme activity assays were performed as previously described by Yahyaa et al.

178 179

31

180

mL GC glass vial containing 10 µM substrate (GPP or FPP), 10 mM MgCl2, 10 µM

181

MnCl2, and assay buffer 50 mM Bis-Tris pH 7.0 in total volume of 100 µL. The reactions

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were incubated for 30 min at 30 ºC. After incubation the samples were analyzed by auto-

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HS-SPME-GC-MS for the identification of volatile terpenes generated during the 30 ºC

184

incubation. As controls, E. coli cells transformed with control plasmids devoid of the

Briefly, 1 to 500 ng of purified recombinant proteins were added into screw-capped 2



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WtDcTPS1 gene, and heat-inactivated WtDcTPS1 protein or assays without GPP/FPP as a

186

substrate were used.

187 188

WtDcTPS1 Transcript Analysis

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For quantitative RT-PCR (qRT-PCR) analysis of WtDcTPS1, total RNA (5 µg)

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from the different tissues (roots, leaves, flowers, fruit pericarp and whole intact fruits) of

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the carrot wild accession 23727 was extracted (SpectrumTM Plant Total RNA Kit, Sigma-

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Aldrich) and reverse transcribed using RevertAid First Strand cDNA Synthesis Kit

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(Thermo Fisher scientific).

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qRT-PCR was performed on an Applied Biosystem StepOnePlus™ Real-Time

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PCR System (Life Technologies) using ABsolute™ Blue qPCR SYBR® Green ROX Mix

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(Tamar Laboratory Supplies LTD, Israel), 5 ng reverse-translated total RNA, and 100 ng

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of

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AAAGATGACACAGCGGGTAAA-3’)

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GCCTCTGAAACCGAAGAAAGA-3’). A relative quantification of gene expression was

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performed using the housekeeping gene tubulin from carrot as a reference gene. The

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primers used for tubulin were: Tubulin_F_qPCR (5’- TCTTGGAGGTGGCACAGGAT-

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3’) and Tubulin_R_qPCR (5’-ACCTTAGGAGACGGGAACACAGA-3’).

each

primers.

Primers

for

WtDcTPS1 and

were

WtDcTPS-qRT-F

WtDcTPS-qRT-R

(5’(5’-

The difference in relative expression levels of WtDcTPS1 was calculated from 2-

203 204

∆∆Ct

205

using at least three biological replicates.

value after normalization of WtDcTPS1 data to tubulin. All analyses were performed

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RESULTS AND DISCUSSION

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Volatile Profiles in Carrot Seeds of Wild Accessions

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We examined the volatile compound composition of freshly harvested tissue from

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different accessions of wild carrot fruits growing in our experimental station. The GC-

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MS analysis of fruits of different accessions of wild carrot indicated that volatile

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compounds in the fruits consist mainly of mono- and sesquiterpene hydrocarbons

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(Supplementary Table S1). There were significant differences in volatile composition

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between different accessions of wild carrot plants. In most, but not all accessions, the

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monoterpene α-pinene was the most abundant volatile, followed by limonene (Figure 1

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and Supplementary Table S1). Bornyl acetate (an oxygenated monoterpene) was found

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to accumulate in high concentration in carrot fruits of wild accession 23727 as compared

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with others investigated in this study (Figure 1 and Supplementary Table S1).

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Interestingly, the carrot fruits of wild accession 23727 show a high accumulation of

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geraniol as a major monoterpene, while it was found only in trace amounts in some fruits,

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or completely absent in others (Figure 2 and Supplementary Table S1). Geraniol has

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been found to accumulate in trace amounts in the essential oil from wild carrot umbels

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(Daucus carota L. ssp. carota) growing in Poland and in Lithuania,16, 20 and in the umbel

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of Daucus muricatus L. from Algeria.30 Geraniol occurs widely in plants, where it

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performs important ecological functions such as to repelling insects, and antimicrobial

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activity.24, 35-38 Industrially, geraniol is widely used in hygiene products, food flavor and

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fragrance.35, 39

229

Wild carrot fruits from Poland contained α-pinene as a main compound in the

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essential oil,20, 40 while wild carrot fruits from Lithuania contained α-pinene as a second



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major compound.16 α-Pinene is one of the most common volatiles in nature, released by a

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wide range of plant species, including coniferous trees, rosemary, lavender and

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turpentine.41 There are many properties, such as fungicidal, bactericidal, and insecticidal

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that has rendered α-pinene a chemical of general use.41 Also, in insects α-pinene serves as

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pheromone precursor in pheromone biosynthesis of scolitids.42

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Many sesquiterpene hydrocarbons were presented, but their total volatile

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composition (ng g-1 FW) was considerably lower than for monoterpenes (Supplementary

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Table S1). It was also noteworthy that the seeds of the three accessions 23727, 20497,

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and 20465 contain high levels of (E)-β-caryophyllene, and α-humulene (Figure 3 and

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Supplementary Table S1). (E)-β-Caryophyllene has been found as a main sesquiterpene

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in wild carrot fruits from Lithuania.16 It has been reported that (E)-β-caryophyllene

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occurs widely in plants, where it performs important ecological functions, for example it

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is emitted by maize (Zea mays) leaves in response to attack by the lepidopteran larvae

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Spodoptera littoralis and released from roots after damage by larvae of the coleopteran

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Diabrotica virgifera.43 However, terpene oil produced in carrot seed tissues is assumed to

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have a wide variety of functions, and also to contribute significantly to fruit and seed

247

aroma and flavor.10, 12, 39

248

Furthermore, some fatty-acid derived volatiles such as cis-3-nonene and octanal

249

were found to accumulate differently in the wild carrot fruits investigated in this study

250

(Supplementary Table S2). Several phenylpropanoid volatiles such as benzaldehyde,

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phenyl ethyl alcohol, cumin aldehyde, eugenol, methyleugenol and E-methylisoeugenol

252

were also observed (Supplementary Table S2). The norisoprenoid volatile 6-methyl-5-

253

hepten-2-one (MHO) as a breakdown product of lycopene was present only in accessions


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20985, and 20497 of D. broteri Ten. (Supplementary Table S2). MHO is also an

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important flavor and aroma volatile found in a number of fruits like tomato.44 These differences of volatile composition of Daucus seeds are similar to those

256 257

reported for other wild carrots species from different countries.11,

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many factors, such as genotypic differences, the stage of development, the environment,

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and the geographical origin, that can considerably influence the volatile composition

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pattern of D. carota.14,

261

different authors and listed in Supplementary Table S1 and S2 are inconsistent to some

262

extent.

16, 17, 27, 40

14, 16, 45, 46

There are

It is therefore not surprising that values obtained by

263 264

Monoterpene and Sesquiterpene Biosynthetic Capacity of Carrot Fruits of Wild

265

Accessions

266

The bulk of mono-, and sesquiterpenes in carrot fruits of wild accessions are

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summarized in Supplementary Table S1. It was therefore of interest to determine TPS

268

activity in the fruit pericarp. Cell-free extracts (protein crude extracts) derived from fruit

269

pericarp were tested for terpene synthase activity. Our results clearly indicate that terpene

270

synthase enzymatic activity is measurable in fruit pericarp. GC-MS analysis of the

271

reaction products catalyzed by the cell-free extracts with GPP as a substrate identified at

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least 11 monoterpenes, with limonene, sabinene, and geraniol as three major products

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(peak #8, #3, and #11, respectively, in Figure 4A), along with α-thujene, α-pinene, β-

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pinene, β-myrcene, α-phellandrene, α-terpinene, γ-terpinene, and α-terpinolene (Figure

275

4A). While incubation of cell-free extracts with FPP led to the production of

276

sesquiterpenes, including daucene, β-bisabolene, and δ-cadinene as the predominate



277

products, along with E-β-caryophellene, E-β-farnesene, and α-humelene (Figure 5A).

278

Heat-denatured cell-free extracts preparations or assays without GPP/FPP as substrates

279

were used as controls for terpenes formation, and no terpene products were observed in

280

these assays (Figure 4B and 5B). The products generated by cell-free extracts from

281

carrot fruit pericarp of wild accession 23727 were the same as those present in these

282

fruits and included the monoterpenes geraniol, sabinene, limonene, α-pinene, β-pinene, β-

283

myrcene, α-phellandrene, and the sesquiterpenes E-β-caryophellene, E-β-farnesene, and

284

α-humelene (Supplementary Table S2).

285 286

These results prompted us to clone and isolate the terpene synthase gene from wild carrot fruits that might be involved in the formation of terpene volatile compounds.

287 288

Isolation of Terpene Synthase Genes from Wild Carrot Fruits

289

To identify terpene synthases sequences from wild carrot fruits we constructed

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degenerated primers based on known TPS genes from other previously characterized

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angiosperm terpene synthases,33 including D. carota DcTPSs.2, 27 Using these primers we

292

could amplify DNA fragment about 300 bp in length. Sequence of this fragment revealed

293

a partial TPS sequence. The complete open reading frame (ORF) of this TPS gene was

294

obtained by 5’-RACE and 3’-RACE. The ORF with 1779 bp was designated as

295

WtDcTPS1. The protein encoding gene WtDcTPS1 exhibited highly conserved sequences

296

of plant TPSs including the asparate-rich motif DDxxD and the RxR motif, both of which

297

are involved in catalysis.29, 47 Also, a RR(x)8W motif is present in the N-terminal region

298

of WtDcTPS1 (Supplemental Figure S1). The motif is assumed to participate in the

299

ionization of the substrate48 and is characteristic of most TPS members of the subfamilies


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TPS-a and TPS-b.49,

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database revealed that the WtDcTPS1 matched the protein sequence encoded by the

302

previously characterized DcTPS2 from D. carota with 97% identity27 (Supplemental

303

Figure S1). Also, the WtDcTPS1 sequence displayed more than 80% identity to several

304

other (recently published) predicted monoterpene syntheses from D. carota

305

(Supplemental Figure S2 and Figure 5).2

306

50

A BLASTP analysis with the WtDcTPS1 in the NCBI protein

However, given the fact that carrot domestication occurred only ~ 1000 years ago

307

and that carrot is an outcrossing species,7,

8

308

between wild and cultivated carrot occurred throughout the history of this crop.2, 7, 8 Also,

309

gene transfer from wild to cultivated plants has contributed to the evolution of several

310

crop species.2, 8

it is likely that bidirectional gene flow

311

A phylogenetic tree based on protein sequence comparisons with representative

312

TPS sequences from other plant species, including several predicted monoterpene

313

syntheses from D. carota 2indicated that the WtDcTPS1 protein belongs to the TPS-b

314

clade (Figure 5), in which the majority of angiospermous monoterpene synthases

315

reside.28 The TPS sequences most similar to WtDcTPS1 are those of a limonene synthase

316

(63% identity) from Eleutherococcus trifoliatus, β-bisabolene synthase-like (59%

317

identity) from Jatropha curcas, monoterpene synthase (55% identity) from Ricinus

318

communis, monoterpene synthase (47 % identity) from Eucalyptus grandis, α-thujene

319

synthase/sabinene synthase (43% identity) from Litsea cubeba and α-phellandrene

320

synthase from Vitis vinifera.

321 322



323

Functional Characterization of the Enzymes Encoded by WtDcTPS1

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E. coli BL21 (DE3) cells expressing His-tagged WtDcTPS1 using the expression

325

vector pEXP5-CT/TOPO TA were harvested and lysed. WtDcTPS1 was purified with a

326

nickel-agarose affinity column to a purity of 90% (data not shown).

327

Upon incubation with GPP as a substrate, WtDcTPS1 catalyzed the formation of

328

mostly geraniol, along with β-myrcene, as detected by gas chromatography-mass

329

spectrometry (GC-MS) analysis (Figure 7A).

330

The identities of these peaks were confirmed by comparisons with retention

331

indices and the mass spectra of authenticity standards. Based on the major product,

332

WtDcTPS1 was designated as a geraniol synthase. Extracts prepared from E. coli (same

333

strain) transformed with pEXP5-CT/TOPO TA lacking a cDNA inserts and heat-

334

denatured enzyme preparations served as controls for terpenes formation independent of

335

WtDcTPS1, and no terpene products were observed in these assays (Figure 7B). Also, it

336

has been show that geraniol is one of the major monoterpens that accumulate in carrot

337

fruits of wild accession 23727 investigated in this study (Table 2, Figure 2).

338

Volatile terpenoids have been reported to be synthesized, and accumulated in

339

fruits, seeds and in different tissues of various plant species.24, 27, 31 Volatile compounds

340

have a wide variety of functions, e.g. in plant defense against pathogens, in the attraction

341

of pollinators by floral scent, or in the attraction of seed dispersing animals by the

342

aromas, or flavors of ripening fruits.24, 51, 52 Besides their ecological benefits to plants,

343

certain terpene compounds are widely used by humans as floral fragrances, flavors, or

344

pharmaceuticals, and some monoterpene compounds have been selected for an important

345

quality trait in plant breeding.53


Page 16 of 39

Page 17 of 39

346


So far, the first cDNA encoding geraniol synthase

12

was cloned from the peltate

347

glands of Ocimum basilicum, a geraniol-producing sweet basil and the resulting protein

348

was functionally characterized to convert GPP into geraniol.54 Consequently, several

349

geraniol synthases encoding genes had been cloned and functionally expressed from

350

various plants, as an active research area for plant secondary metabolism.27, 55-59 Recently,

351

a geraniol synthase was cloned from Camphotheca acuminata, a camptothecin-producing

352

plant, and the recombinant GES showed the conversion activity from GPP to geraniol.55

353

Also, GES from Catharanthus roseus, the anticancer vincristine and vinblastine-

354

producing plant, was cloned and the recombinant CrGES catalyzed geraniol generation,

355

as the precursor for terpenoid indole alkaloid biosynthesis.60

356

It has been suggested that plant terpene synthases share a common evolutionary

357

origin based upon their amino acid sequence homology, conserved sequence motifs, and

358

similar reaction mechanism.61-63 The diversity of the TPS family may be due to the

359

repeated duplication of an ancestral gene and divergence by functional and structural

360

specializations.61 In the phylogenetic tree (Figure 6), the WtDcTPS1 are found clustered

361

together with geraniol synthases of several plants. This result is consistent with an earlier

362

assumption that TPSs having diverged during the course of evolution.28 TPS further

363

divergent to other “specialized” TPS-family enzymes during the time course of evolution

364

to produce diverse terpene volatile compounds.28 This divergent evolution of TPS-family

365

enzymes must have been an ancient event as evidenced by the expectance of the TPS

366

enzyme in wild carrot cultivars.

367 368



369

Expression Patterns of WtDcTPS1

370

To study the WtDcTPS1 gene expression in the wild type carrot leaves, flowers,

371

fruit pericarp, roots, and fruits we measured the accumulation of WtDcTPS1 transcript in

372

all these tissues using qRT-PCR (Figure 8). Transcript abundance was highest for the

373

WtDcTPS1 gene in the fruits, whereas the lowest transcript levels were observed in the

374

flowers, fruit pericarp and in the roots. The expression level of the WtDcTPS1 gene in the

375

fruits of the wild type carrot accession 23727 was 40-fold higher than that found in the

376

roots, leaves, flowers and in the fruit pericarp (Figure 8), which is consistent with the

377

accumulation of high amounts of geraniol found in these fruits (Figure 2 and

378

Supplementary Table S1).

379

A comparison of the expression of the WtDcTPS1 gene in the fruits of wild carrot

380

and the correlation with fruit volatile organic compounds helped us partially to improve

381

our understanding of the regulation of monoterpene synthesis in wild carrot fruits.

382

Furthermore, it is likely that the genome of Daucus carota ssp. carota contains additional

383

TPS genes not identified in RACE-similarity-based cloning strategy. Because terpene

384

volatile compounds may be made by more than one TPS in the same species, and because

385

they accumulate to high levels in such organs, such as fruits, it is generally difficult to

386

determine the direct contribution of each TPS gene to the observed mixture even when

387

the expression levels of the individual TPSs are examined in detail.52.

388

In conclusion, several fruit terpene volatile compounds have been identified by

389

auto-HS-SPME-GC-MS, and the total volatile terpene content varied widely among

390

different carrot fruits of wild accessions. One terpene synthase from carrot fruits of wild

391

accession 23727 (WtDcTPS1) was identified in this study that is involved in geraniol and


Page 18 of 39

Page 19 of 39


392

β-myrcene formation in these fruits. The terpene products found for WtDcTPS1 represent

393

only a fraction of the terpene constituents detected in carrot fruits of wild accessions. The

394

identification of WtDcTPS1 cDNA from wild carrot fruits will facilitate the cloning of

395

additional genes of the TPS family in wild carrot and will allow future molecular and

396

molecular, physiological and biochemical studies of the regulation of flavor and aroma

397

formation in carrot fruits. The WtDcTPS1 gene described here and potential other TPS

398

genes from wild carrot may be developed into molecular markers to aid in breeding and

399

improvement of cultivars with superior fruit carrot flavor and aroma with pharmaceutical

400

interest.

401 402

ABBREVIATIONS USED

403

Auto-HS-SPME-GC-MS:

404

chromatography mass spectrometry; FPP: farnesyl pyrophosphate; GES: geraniol

405

synthase; GGPP: geranylgeranyl pyrophosphate; GPP: geranyl pyrophosphate; MHO: 6-

406

methyl-5-hepten-2-one; TPS: terpene synthase; RACE: Rapid amplification of 5’- and 3’-

407

cDNA ends WtDcTPS1: wild-type Daucus carota L. ssp. carota terpene synthase.

Auto-headspace-solid

phase

micro

extraction

gas

408 409

ACKNOWLEDGEMENTS

410

This research was supported by the Israel Plant Gene Bank under grant [Grant No. 261-

411

1122-14 to Mwafaq Ibdah]. The authors are grateful to Professor Eran Pichersky for

412

critical reading of the manuscript and many discussions during the preparation of this

413

work. We thank Eric Palevsky (Newe Yaar Research Center, Israel) for a final

414

proofreading of the manuscript.



415

Supporting Information

416

The Supporting Information is available free of charge on the ACS Publications website.

417 418

Supplemental Figure S1. Comparison of deduced amino acid sequences of Daucus

419

carota DcTPS2

420

acids not identical in both proteins are marked by black boxes. The highly conserved

421

regions are labeled RRx8W, RxR, and DDxxD.

31

and wild carrot (Daucus carota L. ssp. carota) WtDcTPS1. Amino

422 423

Supplemental Figure S2. Comparison of deduced amino acid sequences of Daucus

424

carota TPSs 2 and wild carrot (Daucus carota L. ssp. carota) WtDcTPS1 from this study.

425 426

Supplemental Table S1. Auto-HS-SPME-GC-MS quantification of mono- and

427

sesquiterpene volatile compounds in nine carrot fruits of wild accessions.

428 429

Supplemental Table S2. Auto-HS-SPME-GC-MS quantification of other volatile

430

compounds of nine carrot fruits of wild accessions.

431 432 433 434 435 436 437


Page 20 of 39

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622 623 624

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625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642


Page 26 of 39

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643


FIGURE CAPTIONS

644 645

Figure 1. The three most abundant monoterpenes in carrot fruits of wild accessions. All

646

analyses were performed using five biological replicates.

647 648

Figure 2. The level of the geraniol in carrot fruits of wild accessions. All analyses were

649

performed using five biological replicates.

650 651

Figure 3. The three most abundant sesquiterpenes in carrot fruits of wild accessions. All

652

analyses were performed using five biological replicates.

653 654

Figure 4. Monoterpene synthase activity in cell-free extracts derived from carrot fruits.

655

(A) Analysis of products of the reaction catalyzed by cell-free extracts with GPP as the

656

substrate. (B) Analysis of products of the reaction catalyzed by boiled cell-free extracts

657

with GPP as the substrate. Identification of the products was done by GC-MS according

658

to the retention index and by comparing the compound mass spectrum to National

659

Institute of Standards and Technology database.

660 661

Figure 5. Sesquiterpene synthase activity in cell-free extracts derived from carrot fruits.

662

(A) Analysis of products of the reaction catalyzed by cell-free extracts with FPP as the

663

substrate. (B) Analysis of products of the reaction catalyzed by boiled cell-free extracts

664

with FPP as the substrate. Identification of the products was done by GC-MS according



Page 28 of 39

665

to the retention index and by comparing the compound mass spectrum to National

666

Institute of Standards and Technology database.

667 668

Figure 6. An unrooted neighbor-joining tree based on protein sequences of WtDcTPS1,

669

and selected plant TPS sequences, including several predicted monoterpene synthases

670

from D. carota

671

was generated using Phylogeny Analysis MEGA 6 program .34 The resulting tree was

672

bootstrap analyzed with 1,000 replicates. The subdivisions (clades) of the TPS gene

673

family, designated TPS-a to TPS-f, are according to Chen et al. (2011). The black bold

674

underline indicates the wild type carrot sequence identified in this study (WtDcTPS1).

675

Ricinus communis (Rc) monoterpene synthase (syn), accession number XM_002515643;

676

Jatropha curcas (Jc) β-bisabolene syn, [XM_012230898]; Eleutherococcus trifoliatus

677

(Et)

678

syn,[XM_01005347]; Litsea cubeba (Lc) α-thujene syn, [HQ651180]; Vitis vinifera (Vv)

679

α-phellandrene syn, [HM807382]; Camptotheca acuminata (Ca) geraniol synthase

680

[KT633829]; Ocimum basilicum (Ob) [AY362553]; Catharanthus roseus (Ca) GES

681

[JN882024]; Valeriana officinalis

682

[DQ234298]; Cinnamomum tenuipilum (Ct) GES [AJ457070]; Abies grandis (Ag) β-

683

phellandrene

684

[AAF61455]; Zea mays (Zm) (E)-β-caryophyllene syn, [ABY79207]; Oryza sativa (Os)

685

(E)-β-caryophyllene/β-elemene syn, [ABJ16553]; Oryza sativa (Os) farnesol syn,

686

[ABJ16554]; Artemisia annua (Aa) β-caryophyllene syn, [AAL79181]; Antirrhinum

687

majus myrcene syn, [AAO41726]; Clarkia breweri (Cb) linalool syn 2 (LIS2);

2

and the previously characterized DcTPS2 from D. carota.

limonene

syn,

syn,

[KJ126717];

[AAF61453];

Eucalyptus

grandis

(Eg)

31

The tree

monoterpene

GES [KF951406]; Perilla citriodora

Abies

grandis

(Ab)


limonene/α-pinene

GES

syn,

Page 29 of 39


688

[AAD19840]; Clarkia breweri (Cb) linalool syn, [AAD19839]; Populus trichocarpa (Pt)

689

copalyl diphosphate syn, [XP_002306777]; Triticum aestivum (Ta) ent-copalyl

690

diphosphate syn, [BAH56558]; Picea glauca (Pg) ent-kaurene syn, [ACY25275];

691

Solanum lycopersicum (Sl) ent-kaurene syn, [AEP82778], and Daucus carota TPS

692

predicted monoterpene synthases, DCAR. 2

693 694

Figure 7. GC-MS of the products generated in vitro by recombinant WtDcTPS1 protein.

695

(A) Analysis of products of the reaction catalyzed by recombinant WtDcTPS1 with GPP

696

as the substrate. (B) Analysis of products of the reaction catalyzed by boiled recombinant

697

WtDcTPS1 with GPP as the substrate. Mass spectrum of: (C) the enzymatic reaction

698

products identified as geraniol and (D) β-myrcene. The insert shows the structures of

699

geraniol and β-myrcene. Identification of other products was done by GC-MS according

700

to the retention index and by comparing the compound mass spectrum to NIST database.

701 702

Figure 8. qRT-PCR determination of transcript levels of WtDcTPS1 in leaves, roots,

703

flowers, fruit pericarp, and fruits (whole intact fruits) of wild carrot accession 23727.

704

Quantification of WtDcTPS1 transcript level by real-time RT-PCR analysis normalized to

705

equal levels of tubulin transcripts. All analyses were performed using three biological

706

replicates.

707 708 709 710



Table 1. Summary of Daucus carota L. ssp. carota plant material used in this study.

Accession numbers 20465 20985 20497 23727 21793 21424 21375 20726 20873

Carrot species Daucus carota L. Daucus broteri Ten. Daucus broteri Ten. Daucus broteri Ten. Daucus glaber (Forssk.) Thell. Daucus glaber (Forssk.) Thell. Daucus glaber (Forssk.) Thell. Daucus aureus Desf. Daucus aureus Desf.

Geographic Region North Golan Shefela Lower Galilee Samaria Mountains Akko Plain Palestine Plain Sharon Plain Sharon Plain Shefela


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Figure 1

Concentration (ng g-1 FW)

1200 1000

α-Pinene Limonene Bornyl acetate

800 600 400 200 0 20465 20985 20497 23727 21793 21424 21375 20726 20873 Wild carrot accessions



Figure 2


7000

Geraniol

6000 5000 4000 3000 2000 1000 0 20465 20985 20497 23727 21793 21424 21375 20726 20873 Wild carrot accessions


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Figure 3


3000 2500

E-β-Caryophyllene α-Humulene γ-Cadinene

2000 1500 1000 500 0 20465 20985 20497 23727 21793 21424 21375 20726 20873 Wild carrot accessions



Figure 4


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Figure 5



Figure 6


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Figure 7



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Relative expression levels

Figure 8

45 40 35 30 25 20 15 10 5 0

WtDcTPS1

Roots

Leaves

Flowers

Fruit pericarp


Fruits

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TOC

Daucus carota L. ssp. carota TPS WtDcTPS1 GPP

OH


Daucus carota L. ssp. carota - ACS Publications - American Chemical

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