Bioactive C17-Polyacetylenes in Carrots (Daucus ... - ACS Publications

Subscriber access provided by UNIV OF WINNIPEG

Review

Bioactive C17-Polyacetylenes in Carrots (Daucus carota L.): Current Knowledge and Future Perspectives Corinna Dawid, Frank Dunemann, Wilfried Schwab, Thomas Nothnagel, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04357 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 19, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

Journal of Agricultural and Food Chemistry

1

Bioactive C17-Polyacetylenes in Carrots (Daucus carota L.):

2

Current Knowledge and Future Perspectives

3 4

Corinna Dawidǂ≠, Frank Dunemann§≠, Wilfried Schwab#, Thomas

5

Nothnagel§, and Thomas Hofmannǂ*

6 ǂ

7

Chair for Food Chemistry and Molecular Sensory Science, Technische Universität

8

München, Lise-Meitner-Straße 34, D-85354 Freising, Germany §

9

Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute

10

for Breeding Research on Horticultural Crops, Erwin-Baur-Strasse 27, D-06484

11

Quedlinburg, Germany #

12

Biotechnology of Natural Products, Technische Universität München, Liesel-

13

Beckmann-Strasse 1, D-85354 Freising, Germany

14 ≠

15

These authors contributed equally to this work.

16 17

Running Title: Bioactive polyacetylenes in carrots.

18 19

*

20

PHONE

+49-8161/71-2902

21

FAX

+49-8161/71-2949

22

E-MAIL

[email protected]

Author to whom correspondence should be addressed

23

ACS Paragon Plus Environment

1


Page 2 of 41

24

ABSTRACT

25

C17-polyacetylenes are a prominent group of oxylipins and are primarily produced by

26

plants of the families Apiaceae, Araliaceae, and Asteraceae, respectively. Recent

27

studies on the biological activity of PAs have indicated their potential to improve

28

human health due to anticancer, antifungal, antibacterial, anti-inflammatory, and

29

serotogenic effects. These findings suggest targeting vegetables with elevated levels

30

of bisacetylenic oxylipins, such as, e.g. falcarinol by breeding studies. Due to the

31

abundant availability, high diversity of cultivars, world-wide experience and its high

32

agricultural yields, in particular, carrot (Daucus carota L.) genotypes are a very

33

promising target vegetable. This article provides a review on falcarinol-type C17-

34

polyacetylenes in carrots and a perspective on their potential as a future contributor

35

to improving human health and well-being.

36 37

KEYWORDS: carrot, bioactive polyacetylenes, falcarinol, falcarindiol, human health,

38

bitterness, Daucus carota L.

39


2

Page 3 of 41

40


INTRODUCTION

41 42

Polyacetylenes (PAs) are a large group of non-volatile bioactive phytochemicals that

43

comprise at least two, usually conjugated, triple carbon-carbon bonds. They are

44

primarily produced by higher plants of the families Apiaceae and Araliaceae both

45

belonging to the order Apiales, but they are also widespread in Asteraceae family

46

members.1,2 Among the most common PAs are falcarinol (1, Figure 1) and

47

falcarindiol (2), which are found in the edible parts of ordinary vegetables and herbs

48

of the Apiaceae family including but not limited to carrots (Daucus carota L.), parsnip

49

(Pastinaca sativa L.), fennel (Foeniculum vulgare (L.) Mill.), celery (Apium graveolens

50

L.), and parsley (Petroselinum crispum (Mill.) Nym.).3 A wide range of bioactivities

51

have been reported for falcarinol-type polyacetylenes including bitter taste,

52

allergenic, antibacterial, antimycobacterial, and antifungal activities.1,2,4,5 Moreover,

53

scientific evidence is mounting that these oxylipins exhibit anti-cancer and anti-

54

inflammatory properties at nontoxic concentrations for humans.1-3

55

In case further in vivo studies will verify the positive health effects of falcarinol-

56

type PAs, breeding studies targeting vegetables with elevated levels of bisacetylenic

57

oxylipins will be necessary. Since their cultivars are grown worldwide, offer a high

58

diversity and provide superior agricultural yields, carrot (Daucus carota L.) genotypes

59

are considered a most promising target vegetable. Therefore, this review

60

summarizes the knowledge on the chemical structures, biosynthesis, genetics, and

61

distribution of falcarinol-type C17-polyacetylenes in carrots and gives a perspective on

62

future knowledge-based carrot breeding programs aimed at elevating the

63

concentration of polyacetylenes in specific carrot cultivars holding potential to

64

contribute to human health management.

65 ACS Paragon Plus Environment

3


66

Page 4 of 41

CARROTS AND THEIR POLYACETYLENES

67 68

Because of its high yield potential and use as fresh or processed product, cultivated

69

carrot (Daucus carota ssp. sativus Hoffm.) is one of the most important vegetable

70

plants in the world.6 With a current annual world production of more than 30 million

71

tons and a total growing area of about 1.5 million hectares,7 carrots rank among the

72

top ten vegetable crops with Unites States, China, and Russia accounting for 34% of

73

the global production. Carrot is the most widely grown species of the genus Daucus,

74

a member of the large and complex Apiaceae family. Already in the early 18th century

75

Henri Vilmorin started intensive selection breeding with carrots in France. Today,

76

enormous experience exists in carrot breeding, which changed in the last century

77

from mass selection to open pollinated F1-hybrid breeding.6 Sophisticated breeding

78

experiments offer the possibility to improve abiotic and biotic stress tolerance as well

79

as sensory quality of carrots, and to develop genotypes enriched in selected

80

secondary metabolites such as β-carotene.6 In comparison to the breeding

81

achievements, the molecular knowledge including genetic data about carrot traits is

82

much younger and was rather limited until recently. The small haploid genome size of

83

473 Mbp, which is in the same range as found for rice,8 greatly facilitated detailed

84

molecular and genomic studies in carrots. Carrot linkage maps have been developed

85

based on several types of molecular markers.9-11 Genetic diversity of the genus

86

Daucus has been intensively studied through polymorphic DNA markers.12,15 Several

87

carrot transcriptomes and a first de novo assembled whole-genome sequence have

88

been revealed by next generation sequencing (NGS) technology.13,14 Moreover,

89

carrot is well-known as a model species for gene transfer using both genetic

90

modifications by vector and non-vector methods, which is a major prerequisite for

91

functional gene studies.16 ACS Paragon Plus Environment

4

Page 5 of 41


92

Bisacetylenic Oxylipins in Carrots. Although first phytochemicals have been

93

analyzed in carrots already more than hundred years ago, polyacetylenic oxylipins

94

have been under investigation since the middle of the last century. Special interest

95

has been focused on the polyacetylenes´ chemical structures and biological

96

activities. Among the more than 1400 polyacetylenes reported in higher plants,3 a

97

subset of 12 structurally related bisacetylenic oxylipins were isolated from Daucus

98

carota, purified, and their chemical structures identified (Figure 1). Already in 1969,

99

the aliphatic polyacetylenes falcarinol (1), falcarindiol (2), and falcarindiol-3-acetate

100

(3) were isolated from carrots, sharing two double bonds at position C1/2 and C9/10,

101

two triple carbon-carbon bonds at position C4/5 and C6/7, as well as an aliphatic C7-

102

residue (C11-C17).17 While falcarinol (1) exclusively features one hydroxyl function at

103

position C3, falcarindiol (2) has a second hydroxyl group at position C8. Compared to

104

2, falcarindiol-3-acetate exhibits a further acetylic residue at position C3 (3). Besides

105

these quantitatively predominating polyacetylenes, nine additional bisacetylenes

106

have been recently identified in Daucus carota, namely (E)-isofalcarinolone (4),

107

falcarindiol-8-acetate (5), 1,2-dihydrofalcarindiol-3-acetate (6), (E)-falcarindiolone-8-

108

acetate (7), (E)-falcarindiolone-9-acetate (8), 1,2-dihydrofalcarindiol (9), (E)-1-

109

methoxy-falcarindiolone-8-acetate (10), (E)-1-methoxy-falcarindiolone-9-acetate (11),

110

and panaxydiol (12, Figure 1).18,19 Among the bisacetylenes, compounds 4, 6-8, 10,

111

and 11 were reported for the first time in literature and compounds 5, 9, and 12 have

112

previously not been reported as phytochemicals in carrots, e.g. falcarindiol-8-acetate

113

(5) was isolated from Angelica japonica A. Gray and Centella species,20,21 and 1,2-

114

dihydrofalcarindiol (9) and panaxydiol (12) from devil´s club (Oplopanax horridus

115

(SM.) MIg.), ginseng (Panax ginseng C. A. Mey), fennel, and parsley.22-24

116

Unequivocal structure determination of the carrot’s C17-polyacetylenes was

117

possible by means of UV-Vis measurements, LC-MS/MS, LC-TOF-MS, 1D/2D-NMR ACS Paragon Plus Environment

5


Page 6 of 41

118

spectroscopic experiments, as well as co-chromatography with the synthesized

119

reference compounds have been performed in the past (cf. Supporting Information

120

Table S1-3).4,18,19 In particular, the

121

atoms C4–C7 resonating between 64.0 and 84.8 ppm have been found to be of

122

prime importance in establishing the characteristic triple carbon-carbon bonds in the

123

PAs

124

polyacetylenes, including acetylation at position 3 or 8 as found in compounds 3, 5-8,

125

10, and 11, or methoxylation at carbon C18 as found in compounds 10 and 11, can

126

easily be assigned by comparing 1H and

127

revealed typical chemical

128

(CH3), methoxylation could be easily confirmed by resonance signals at 58.1 ppm in

129

the 13C NMR spectrum and 3.35 ppm in the 1H NMR spectrum. These marker signals

130

helped to elucidate the chemical structure of the PAs by means of sophisticated 2D-

131

NMR experiments like COSY, HMQC, or HMBC (cf. Supporting Information Table

132

S1-3).

(Supporting

Information

13

13

C chemical shifts of the quaternary carbon

Table

Structural

S2).

modifications

of

C17-

13

C data. While all the acetyl groups

C shifts centering around 170 ppm (C=O) and 22 ppm

133

As the absolute configuration of falcarindiol (2) has been found to be strongly

134

dependent on the botanical source,22,24-27 several attempts have been made to

135

prepare all possible stereoisomers by means of enantioselective synthesis.18,28-30.

136

For an unequivocal determination of the configuration of 2 from carrots, Schmiech

137

and coworkers developed a novel enantioselective 10-step total synthesis involving a

138

Cadiot-Chodkiewicz cross-coupling reaction of (S)- and (R)-trimethylsilanyl-4-

139

dodecen-1-yn-3-ol and (R)- and (S)-5-bromo-1-penten-4-yn-3-ol, respectively, to

140

generate all possible stereoisomers, namely (3R,8R)-, (3R,8S)-, (3S,8R)-, and

141

(3S,8S)-falcarindiol (Figure 2).18 Comparative chiral HPLC analysis of the synthetic

142

stereoisomers with falcarindiol (2) isolated from carrot extracts led to the unequivocal

143

assignment of the (3R,8S)-configuration in carrots.18 ACS Paragon Plus Environment

6

Page 7 of 41


144

Quantification and Distribution of Polyacetylenes in Daucus carota. An

145

important aspect of the modern use of plant extracts as pharmaceutical preparations

146

or food supplements is the reliable molecular characterization of the bioactive

147

constituents. Due to the high instability of C17-polyacetylenes when exposed to light

148

and/or higher temperature, gentle techniques need to be applied for successful

149

chromatographic isolation of reference materials needed to study their bioactivity in

150

cell-based assays or intervention studies.18,31 Similarly, high requirements are

151

provided for accurate quantification of PAs by means of HPLC-UV,32,33 GC–FID or

152

GC–MS,34-36 and LC–MS/MS3,37 in plant extracts and plasma samples. Moreover,

153

FT-Raman spectroscopy has been reported as a non-destructive method to visualize

154

the major PAs in the different tissues within the same plant.38,39

155

The distribution of falcarinol (1), falcarindiol (2), and falcarindiol-3-acetate (3)

156

is known to vary among carrot cultivars; especially, a major difference can be seen

157

between cultivated orange carrots and the wild relative D. carota ssp. maritimus

158

(Lam.) Batt.40 Although previous literature reports focus mainly on quantification of

159

falcarinol (1), falcarindiol (2) and falcarindiol-3-acetate (3), most of the authors

160

assume that general contents of PAs depend on factors such as cultivar, age,

161

physiological stage, root size, storage time, and geographical location of carrots.

162

Moreover, biotic and abiotic stress factors during growth in the field as well as during

163

post-harvest storage were reported to influence the levels of PAs in carrots.32,34,41-47

164

According to Czepa and Hofmann,34 the most abundant PA in cultivated orange

165

carrots (D. c. ssp. sativus) is falcarindiol with a concentration range from 16 to 84

166

mg/kg fresh weight (FW), followed by falcarinol (8-27 mg/kg FW) and falcarindiol-3-

167

acetate (8-40 mg/kg FW). In particular, the genotype seems to influence the amounts

168

of falcarinol and its analogues,34,48,49 e.g. analysis of falcarinol in 27 different carrot

169

cultivars grown and harvested under the same conditions revealed concentrations of ACS Paragon Plus Environment

7


Page 8 of 41

170

2 ranging from 7.0 to 40.6 mg/ kg FW. Compared to cultured forms of carrots, the

171

levels of 1-3 in some D. carota wild relatives, such as, e.g. D. c. ssp. maximus Desf.,

172

D. c. ssp. maritimus, or D. c. ssp. halophilus, can be up to 10 - 20 times higher

173

(Table 1).38,49-51 Moreover, comparative quantification of 1-3 in 100 genotypes of

174

cultivated carrots and 104 genotypes of wild carrots revealed a large variation for the

175

PA contents between the tested genotypes, especially for the wild relatives (Figure

176

3).49 While the levels of falcarinol (1) varied extremely in the carrot wild relatives

177

ranging from 0.1 to 148.2 mg/100 g FW, the PAs 2 and 3 reached a maximum

178

concentration of 568.4 and 51.7 mg/100 g FW in wild carrots, respectively. In

179

comparison, the cultivars contained explicitly lower concentrations of PAs ranging

180

from 0.08 to 28.1 (1), 0.8 to 42.4 (2), and 0.1 to 14.9 mg/100 g FW (3), respectively.

181

In addition, the ratio of those three PAs (1-3) varies considerably from variety to

182

variety. For example, while in D. c. ssp. sativus cv. Anthonia the ratio from compound

183

no. 1 to 2 to 3 is 2:3:1, in D. c. ssp. sativus cv. Yellowstone the ratio from compound

184

no. 1 to 2 to 3 is 2:11:2. The lack of a good correlation between the PAs (Table 1 and

185

Figure 3) just underlines the importance of a versatile analytical method determining

186

the contents of each individual PA separately.

187

Besides varietal differences, the PA distribution among the organs of carrots

188

plants varies considerably. For example, Czepa and coworkers analyzed the spatial

189

distribution of 1-3 from the top to the bottom as well as from the outer phloem to the

190

inner xylem of carrot roots.4 While the bitter tasting upper end and the phloem

191

contained 33.5 and 32.3 mg/kg FW of 3, significantly lower concentrations of 1.8 and

192

1.5 mg/kg FW were found in the less bitter lower end and the xylem. Among the

193

group of bisacetylenic oxylipins, falcarinol (1) and falcarindiol (2) showed a different

194

spatial distribution than falcarindiol-3-acetate (3); the concentrations of 1 and 2 in the

195

phloem equaled those found in the xylem, whereas the content of 3 in the phloem ACS Paragon Plus Environment

8

Page 9 of 41


196

was double the amount determined in the xylem. By means of in situ Raman

197

mapping experiments the PAs were proposed to be located in vascular bundles in a

198

young secondary phloem as well as in pericycle oil channels in the vicinity of the

199

periderm, which could be responsible for the transport and accumulation of

200

polyacetylenes.40 Moreover, analysis of the PA distribution in roots of carrot wild

201

species, namely D. carota ssp. gummifer Hook. F., D. c. ssp. commutatus Paol., and

202

D. c. ssp. halophilus Brot showed that the whole phloem tissue seems to be rich in

203

polyacetylenes with a maximum to be observed near the pericyclic parenchyma.40

204

The localization of PAs in exterior tissue layers is consistent with their general role in

205

providing an antifungal shield for young roots. These compounds play an important

206

role in plant defense against phytopathogenic fungi, nematodes, and insects,40 e.g.

207

accumulation of polyacetylenes like falcarinol was observed in tomato fruits and

208

leaves attacked with Cladosporium fulvum Cooke, Verticillium albo-atrum Reinke &

209

Berth., and Fusarium oxysporum Schlecht.52

210

fungal leaf blight pathogen Alternaria dauci was shown to be relatively strongly

211

inhibited by falcarindiol. The falcarindiol levels measured in leaves of the partially

212

resistant cultivar 'Bolero' were suggested to be sufficient to inhibit A. dauci growth.53

213

In consequence, PAs are considered to be phytoalexins, low molecular weight

214

compounds produced by plants to respond to microbial attack or abiotic stress such

215

as UV irradiation or salt stress.52

In carrots, the development of the

216

Taking all these findings together, the PA contents in carrots show an

217

enormous genotypic variability in the genus Daucus. The direct utilization of Daucus

218

wild relatives for commercial PA production by conventional field growing is likely to

219

be rather inefficient because of the generally small and branched tap-roots and a low

220

root yield potential of 5 to 10% compared to cultivated carrots (Figure 4). In addition,

221

due to the high PA content the most interesting species and subspecies are native in ACS Paragon Plus Environment

9


Page 10 of 41

222

subtropical climates and need no or just a very low vernalization period. Therefore,

223

flowering occurs early in the vegetation period, which might lead to enhanced

224

lignification of the root tissue, a dramatic reduction of root quality and yield, and a

225

dramatic loss of commercial value.54 The genetic background of flower induction for

226

carrot as well as their wild ancestor is yet not well understood. Recently, only one

227

gene Vern1, that is proposed to control the vernalization requirement, was identified

228

and mapped to chromosome 2, but a number of experiments have shown that bolting

229

is a more complicated trait influenced by much more than the Vern1 gene.55,56

230

Targeted breeding programs for either the selection of high-yield, late bolting wild

231

carrot genotypes or for the creation of PA enriched cultivated carrots are needed for

232

an economically drug production on a field-scale basis.

233

Bitter Taste of Polyacetylenes in Carrots. Apart from 6-methoxymellein,

234

C17-polyacetylenes have been shown to contribute to the undesirable bitter off-taste

235

of certain carrot cultivars and products, such as purees and juices.4,5,34 Due to the

236

fact that the attractive sweet sensory quality of carrots/carrot products is disrupted by

237

a sporadic bitter off-taste, which is often the reason for adverse consumer reactions

238

and causes a major problem for vegetable processors. Czepa et al.4,34 as well as

239

Schmiech et al.5,19 analysed the bitter tasting key phytochemicals by means of a

240

sensomics approach.57 Among other bitter phytochemicals, such as, e.g. 6-

241

methoxymellein, laserin, epilaserin and laserin oxide, in particular, falcarinol (1),

242

falcarindiol (2), and falcarindiol-3-acetate (3) were identified with human recognition

243

threshold concentrations between 40 and 200 µmol/kg. In order to study the

244

importance of these oxylipins as bitter compounds in fresh and processed carrots on

245

the basis of dose/activity relationships, Czepa and Hofmann quantitatively

246

determined their exact concentrations by means of GC-MS.34 On the basis of Dose-

247

over-threshold (DoT) factors, calculated as the ratio of the concentration and the ACS Paragon Plus Environment

10

Page 11 of 41


248

human sensory threshold of a compound, a close relationship between the

249

concentration of falcarindiol (2) and the intensity of the bitter off-taste in carrots,

250

carrot puree, and carrot juice was demonstrated. Furthermore, sensory guided

251

analysis showed that in different carrot segments, such as the peels, concentrations

252

of falcarinol (1) and falcarindiol-3-acetate (3) also could exceed their taste thresholds

253

and directly contribute to the bitterness of carrots.19 Therefore, the PAs 1-3 were

254

suggested as the key markers for an objective evaluation of the taste quality of carrot

255

products and peeling carrots rich in PAs, allows reducing the bitter off-taste to some

256

extend while high falcarinol content is maintained.19,58 Although previous studies

257

gave first insights in carrots bitterness, surprisingly, nothing is known about the

258

compounds which contribute to the enhanced bitterness reported for wild type carrots

259

like D.c.ssp. halophilus or D. c. ssp. maritimus.

260

Several researchers observed a correlation of carrot´s bitter off-flavor to

261

abiotic and biotic stress factors affecting the carrot metabolism during harvesting,

262

transportation, storage and processing.4,5,34,59-64 Particularly, the study of Seljåsen et

263

al.61 illustrates that mechanical stress from field to consumer caused by shaking in a

264

transport simulator at post-harvest directly influences the taste and aroma quality of

265

fresh carrots. Although modern breeding techniques and cultivar selection have been

266

helpful to improve the sensory quality, and high concentrations of falcarinol-type PAs

267

are known to contribute to the bitter off-flavor in vegetables, to date no dose/activity

268

considerations on key phytochemicals of carrots influenced by those different

269

external factors are available.

270 271

BIOSYNTHESIS

AND

272

POLYACETYLENES

MOLECULAR

GENETICS


OF

CARROT

11


Page 12 of 41

273

Despite the extensive research concerning the analytical and biochemical

274

identification and characterization of plant falcarinol-type PAs, and the comparatively

275

large number of reports about their putative biological functions, by far less

276

knowledge exists about the structure and function of the enzymes involved in

277

biosynthesis of falcarinol-type PAs. In addition, the molecular-genetic principles

278

underlying PA production in different plant tissues are poorly understood and nearly

279

nothing is known about the genetics and inheritance of the patterns and

280

concentrations of specific PAs in (crop) plants.

281

Results of metabolic studies have pointed out the important role of crepenynic

282

and dehydrocrepenynic acid as precursors of PAs that are known to occur in plants

283

of Apiaceae, Araliaceae, Asteraceae and of some other species as for example the

284

Solanaceae.52 The crepenynate pathway for acetylenic natural product biosynthesis

285

has been examined repeatedly in plants and fungi over the past 50 years (Figure 5).

286

The pathway is fed with acetate-derived acyl lipids provided from primary metabolism

287

and diverges with the conversion of linoleic acid to crepenynic acid.52 As "unusual"

288

fatty acids, crepenynic and dehydrocrepenynic acids are rarely accumulated in plant

289

tissues, and their function and control mechanisms are poorly explored.65 It is likely

290

that these fatty acids are rapidly metabolized for the formation of secondary bioactive

291

molecules such as falcarinol. In D. carota,

292

shown to be incorporated into falcarinol when provided as an exogenous precursor to

293

the carrot cells.66 Similarly, in Panax ginseng NMR-based isotopologue profiling of

294

panaxydol and panaxynol confirmed their assumed origin from acetyl-CoA/malonyl-

295

CoA via crepenynate as the putative intermediate.67

14

C-labeled crepenynic acid has been

296

Fatty acid desaturases are enzymes capable of modifying pre-existing carbon-

297

carbon bonds within fatty acids. These enzymes vary in specific function and are

298

responsible for the modification of a wide spectrum of fatty acids found throughout ACS Paragon Plus Environment

12

Page 13 of 41


299

nature. They are regioselective, display substrate selectivity, and can introduce

300

functionality in a stereospecific manner. The enzyme primarily responsible for the

301

synthesis of linoleic acid from oleic acid is a ∆12-fatty acid desaturase (FAD2). This

302

microsomal enzyme introduces a double bond at the ∆12-position of oleic acid,

303

forming linoleic acid on the endoplasmic reticulum.68,69 Variants of the FAD2 enzyme

304

are also known to have diversified functionalities in fatty acid modification, catalysing

305

hydroxylation,70,71 epoxidation,72 and the formation of acetylenic bonds and

306

conjugated double bonds.73 Some functionally divergent FAD2 enzymes are multi-

307

functional, such as the bifunctional hydroxylase/desaturase from Lesquerella fendleri

308

(Gay) Watson.74 Diverged FAD2 homologues that introduce a triple bond within a

309

fatty acid are designated as acetylenases.75

310

FAD2 is among the best-studied plant fatty acid desaturase gene families, in

311

terms of both molecular and biochemical investigations. Since the cloning of the first

312

plant FAD2 gene from Arabidopsis thaliana (L.) Heynh.,68 its orthologous DNA

313

sequences have been isolated and characterized from many different plant species,

314

mainly before the background to study the biosynthesis of fatty acids important for

315

seed oil quality.76-80 Only a single FAD2 gene exists in Arabidopsis, but in most other

316

plant species FAD2 is encoded by small gene families. For example, FAD2 is

317

encoded by two distinct FAD2 genes in soybean (Glycine max (L.) Merr)76 and flax

318

(Linum usitatissimum L.),80 three genes in sunflower (Helianthus annuus L.),81 and by

319

five species of the genus Gossypium.78 In safflower (Carthamus tinctorius L.), an

320

ancient oilseed crop from the Asteraceae family, an unusually large FAD2 gene

321

family with 11 members was described.65 In the past years a number of FAD2-

322

divergent genes have been identified in other members of the Asteraceae family

323

such as marigold (Calendula officinalis L.), hawksbeard (Crepis alpina L.) and

324

sunflower (H. annuus)72,82,83 and have been associated with synthesis of divergent ACS Paragon Plus Environment

13


Page 14 of 41

325

fatty acid structures that may play roles in resistance to biotic stresses. The Crep1

326

gene for the acetylenase from C. alpina was the first cloned gene for a functional

327

acetylenase. The FAD2-related enzyme controlled by Crep1 accumulates large

328

amounts of the acetylenic fatty acid crepenynic acid in the seeds of C. alpina.72 The

329

related enzyme of the parsley gene ELI12, which was previously shown to encode

330

also a divergent triple-bond form of FAD2, could be induced by a fungal elicitor.83

331

It is intriguing how the divergent FAD2 gene family members with similar

332

fundamental properties carry out specific functions. It has been demonstrated

333

through site directed mutagenesis, that very few amino acid changes are required to

334

change the enzymatic function of a FAD2 gene. For instance, as few as four amino

335

acid changes in a FAD2 fatty acid desaturase were required in order to obtain

336

hydroxylase activity, and conversely, substitution of six amino acids could convert a

337

fatty acid hydroxylase into a fatty acid desaturase.74 It is suggested that a switch from

338

desaturase to acetylenase might involve more extensive changes in sequence than

339

that required to interchange between a fatty acid desaturase and a fatty acid

340

hydroxylase. The origins of specificity leading to acetylenases and desaturases are

341

not currently evident from comparisons at the primary sequence level, and residues

342

promoting acetylenase activity have yet to be located.65 The question, how exactly

343

compounds like falcarinol or falcarindiol are synthesized in higher plants by FAD2-

344

related acetylenases, has still to be answered. Apiaceae plants are not known to

345

accumulate relevant amounts of crepenynic and dehydrocrepenynic acid (the

346

assumed precursors of falcarinol-type PAs, see Figure 2), but both substances are

347

believed to be intermediates in the biosynthetic pathway of these PAs. Falcarinol

348

would then be rapidly metabolized, perhaps also in response to fungal pathogenesis

349

as supposed in the case of ELI12.83 Exploring and identifying the function and control

350

mechanisms of such cryptic expression of unusual fatty acids in D. carota would be ACS Paragon Plus Environment

14

Page 15 of 41


351

one of the major prerequisites for the development of a molecular breeding system

352

using DNA markers generated from the target genes.

353

If FAD2-related genes for falcarinol-type PA production could be identified,

354

functional markers might be developed for future molecular breeding approaches

355

aimed at carrot cultivars with high or low contents of falcarinol-type PAs, respectively.

356

First of all, a pre-screening of putative crossing parents might be performed by

357

analysing the functional allelic diversity of Daucus germplasm, to select the most

358

promising genotypes. If such genotypes are used for crosses in carrot breeding, the

359

gene-specific markers can be used in MAS (marker assisted selection) to screen the

360

progenies for the desired gene combinations.

361

To identify D. carota FAD2 orthologous sequences, the assembled carrot

362

transcriptome was used for in silico gene mining.13 By using several published plant

363

FAD2 protein sequences as query for BLAST (Basic Local Alignment Search Tool),

364

six putative functional candidate FAD2 genes were identified among the about

365

60,000 carrot EST contigs. They have been preliminary designated as DcFAD2-1 to

366

DcFAD2-6 (Figures 6, A and B). DcFAD2-1 and DcFAD2-2 appear to be transcribed

367

genes in the carrot genome, as shown by reverse transcriptase (RT)-PCR. Their

368

sequences are divergent from typical FAD2s as they belong to the cluster of putative

369

plant acetylenases (Figure 6 A), whereas the other four sequences probably encode

370

desaturases or hydroxylases. DcFAD2-2 is highly similar (amino acid identity 96%) to

371

the parsley gene PcELI12 which is known as an FAD2-derived acetylenase.83

372

Screening the Panax ginseng transcriptome published by Li et al.

373

single contig containing the candidate gene PgFAD2-1, which also might be a

374

putative acetylenase gene (Figure 6, A and B). It has been proposed that the amino

375

acid G (Glycine) immediately preceding the first highly conserved histidine-rich motif

376

(histidine box) might indicate the functionally divergent FAD2s like acetylenases.79 ACS Paragon Plus Environment

84

results in a

15


Page 16 of 41

377

This might be a further indication for the assumption, that DcFAD2-1, DcFAD2-2, and

378

PgFAD2-1 have an acetylenase function. Work is in progress to continue molecular

379

characterization of Daucus FAD2 candidate genes and their putative biochemical

380

functions.

381 382

BIOACTIVITY OF CARROT POLYACETYLENES

383

The list of biological and pharmacological activities associated with PAs is increasing

384

and these diacetylenes are considered to contribute to the health benefits associated

385

with the consumption of fruit and vegetables.85,86 In particular, aliphatic C17-PAs of

386

the falcarinol-type have been shown to exhibit potent anti-microbial, anti-

387

inflammatory, and anti-cancer effects.87 For further review see reports from

388

Christensen and Brandt,3 and Christensen.1,2 A series of in vitro and in vivo studies

389

showed convincing evidence for the cytotoxic and chemopreventive activity of

390

falcarinol, panaxynol and related diacetylenes.2,3,24,37,88 For example, falcarinol,

391

panaxydol and panaxytriol were reported to exhibit high cytotoxic activity to leukemia

392

(L-1210), mouse fibroblast-derived tumor cells (L-929), mouse melanoma (B-16), and

393

human gastric adenocarcinoma (MK-1) cells with lowest ED50 values of 0.108, 0.059,

394

and 0.605 µM, respectively, found in MK-1 cancer cell studies.2,89-91 Interestingly, the

395

ED50 against normal human fibroblast cells (MRC-5) were almost 20 times higher

396

when compared to those of the MK-1 cancer cells, thus indicating that these

397

phytochemicals may be useful in cancer treatment.3,90

398

Only recently, it has been demonstrated that falcarinol-type PAs function as of

the

breast

cancer

resistance

protein

BCRP/ABCG2.87

399

inhibitors

400

BCRP/ABCG2 is an efflux transporter important for xenobiotic absorption and

401

disposition, the results indicate a prospective use of PAs as multidrug resistance

402

reversal agents for cancer chemotherapy.87 A further in vitro study highlighted that ACS Paragon Plus Environment

Since

16

Page 17 of 41


403

falcarinol could stimulate differentiation of primary mammalian cells at low falcarindiol

404

concentrations of 0.001 to 0.1 µg/mL.92 Based on data obtained from in vitro

405

experiments and those from human bioavailability studies, falcarinol-type PAs are

406

considered to exhibit a positive inhibitory effect on cancer cell proliferation.1,2 For

407

example, when falcarinol was administrated orally to humans via carrot juice (0.9 L;

408

13.3 mg falcarinol/L), it was rapidly absorbed and reached a maximum serum level of

409

2.3 and 2.0 ng/mL, respectively, 2 and 5 hours after administration.1 Next to

410

falcarinol, also falcarindiol, panaxydiol, and falcarindiol-8-methyl either were shown to

411

possess cytotoxic effects on human cancer and leukemia cells and anti-mutagenic

412

activity in vitro, although they appear to be less bioactive than falcarinol. Moreover,

413

cell-based assays revealed synergistic effects when falcarinol-type PAs were

414

administered in a cocktail.2 These finding necessitates two types of advanced studies

415

in parallel: On the one hand the health-promoting effects of carrot PAs in vivo have to

416

be confirmed by clinical as well as in further preclinical studies.1 On the other hand

417

the above mentioned results suggest performing breeding studies targeting specific

418

carrot genotypes with elevated PA concentrations.

419 420

CONCLUSION AND FUTURE DIRECTIONS

421

PAs, comprising a group of natural phytochemicals produced by higher plants of the

422

families Apiaceae and Araliaceae, demonstrate a broad range of bioactivities and are

423

believed to contribute to the health benefits associated with the consumption of fruit

424

and vegetables. Thus, breeding of vegetables with elevated bisacetylenic oxylipin

425

concentrations appears to be a worthwhile endeavor. Due to Europe’s climatic

426

conditions and cultivar experience as well as due to the high agricultural yields, in

427

particular, carrot (D. c. ssp. sativus) genotypes look as a very promising target

428

vegetable. Out of the twelve known C17-PAs in carrots falcarinol and falcarindiol have ACS Paragon Plus Environment

17


Page 18 of 41

429

been identified as the major oxylipins and were demonstrated to inhibit the cancer

430

cell growth in in vitro and in vivo studies. Although the present review has

431

demonstrated that PAs have the reputation for improving human health and well-

432

being, the health-promoting effects of carrot PAs in vivo should be confirmed in more

433

detail by clinical as well as in further preclinical studies in future. However, these PAs

434

have also been shown to contribute to the bitter off-taste of certain carrot cultivars

435

and products. Whereas higher concentrations of PAs in Apiaceae vegetables are

436

detrimental for palatability due to their bitter off-taste, the content of PAs in carrots to

437

be used for pharmaceutical purposes need to be high enough to allow for a cost-

438

efficient drug production.

439

Breeding specific carrot chemotypes might efficiently enhance the commercial

440

production of the pharmaceutically relevant PAs, since the direct usage of wild carrot

441

relatives rich for the wanted PAs is recently neither practicable nor economical. For

442

breeding experiments including future approaches of targeted gene editing, the

443

knowledge on the structure and function of enzymes involved in biosynthesis of PAs

444

like falcarinol need to be widened.

445 446 447

LITERATURE CITED

448

(1)

Christensen, L. P. Bioactivity of polyacetylenes in food plants. In: Watson, R. R.;

449

Preedy, VR, Eds. Bioactive foods in promoting health. Oxford: Academic press

450

2010, 285–306.

451

(2)

Christensen, L. P. Aliphatic C17-Polyacetylenes of the falcarinol type as

452

potential health promoting compounds in food plants of the Apiaceae family.

453

Recent Patents on Food, Nutrition & Agriculture 2011, 3, 64–77.


18

Page 19 of 41

454


(3)

Christensen, L.P.; Brandt, K. Bioactive polyacetylenes in food plants of the

455

Apiaceae family: Occurrence, bioactivity and analysis. J. Pharm Biomed. Anal.

456

2006, 41, 683–693.

457

(4)

Czepa, A.; Hofmann, T. Structural and sensory characterization of compounds

458

contributing to the bitter off-taste of carrots (Daucus carota L.) and carrot puree.

459

J. Agric. Food Chem. 2003, 51, 3865–3873.

460

(5)

Schmiech, L.; Uemra, D.; Hofmann, T. Reinvestigation of the bitter compounds

461

in carrots (Daucus carota L.) by using a molecular sensory science approach. J.

462

Agric. Food Chem. 2008, 56, 10252–10260.

463

(6)

464 465

sativus Hoffm.). Plant Breeding 1995, 114, 1–11. (7)

466 467

FAO, Food and Agriculture Organization of the Unitated Nations, Crops. http.//faostat.fao.org/, 2012.

(8)

468 469

Stein, M.; Nothnagel, Th. Some remarks on carrot breeding (Daucus carota

Arumuganathan, K.; Earle, E. D. Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 1991, 9, 208–218.

(9)

Cavagnaro, P. F.; Chung, S.-M.; Manin, S.; Yildiz, M.; Ali, A. Microsatellite

470

isolation and marker development in carrot - genomic distribution, linkage

471

mapping, genetic diversity analysis and marker transferability across Apiaceae.

472

BMC Genomics 2011, 12, 386.

473

(10) Alessandro, M. S.; Galmarini, C. R.; Iorizzo, M.; Simon, P. W. Molecular

474

mapping of vernalization requirement and fertility restoration genes in carrot.

475

Theor. Appl. Genetics 2013, 126, 415–423.

476

(11) Budahn, H.; Baranski, R., Grzebelus, D.; Kiełkowska, A.; Straka, P., Metge, K.;

477

Linke, B.; Nothnagel, T. Mapping genes governing flower architecture and

478

pollen development in a double mutant population of carrot. Front. Plant Sci.

479

Plant Gen. Genomics 2014, 5, 1–10. ACS Paragon Plus Environment

19


Page 20 of 41

480

(12) Baranski, R.; Maksylewicz-Kaul, A.; Nothnagel, T.; Cavagnaro, P. F., Simon, P.

481

W.; Grzebelus, D. Genetic diversity of carrot (Daucus carota L.) cultivars

482

revealed by analysis of SSR loci. Genet. Resour. Crop Evol. 2012, 59, 163–

483

170.

484

(13) Iorizzo, M.; Senalik, D. A.; Grzebelus, D.; Bowman, M.; Cavagnaro, P. F., et al.

485

De novo assembly and characterization of the carrot transcriptome reveals

486

novel genes, new markers, and genetic diversity. BMC Genomics 2011, 12,

487

389.

488 489

(14) Xu, Z.-S.; Tan, H.-W. ; Hou, X.-L.; Xiong, A.-S. CarrotDB: a genomic and transcriptomic database for carrot. Database, 2014, 1-8

490

(15) Grzebelus, D.; Iorizzo, M.; Senalik, D.; Ellison, S.; Cavagnaro, P.; Macko-

491

Podgorni, A.; et al. Diversity, genetic mapping, and signatures of domestication

492

in the carrot (Daucus carota L.) genome, as revealed by diversity arrays

493

technology (DArT) markers. Mol. Breed. 2014, 33, 625–637.

494 495 496

(16) Baranski, R. Genetic transformation of carrot (Daucus carota) and other Apiaceae species. Transgen. Plant J. 2008, 2, 18-38. (17) Bentley, R. K.; Bhattacharjee, D.; Jones, E. R. H.; Thaller, V.

Natural

497

acetylenes. Part XXVIII. C17-polyacetylenic alcohols from the umbellifer

498

Daucus carota L. (carrot): alkylation of benzene by acetylenyl(vinyl)carbinols in

499

the presence of toluene-p-sulphonic acid. J. Chem. Soc. 1969, 685–688.

500

(18) Schmiech, L.; Alayrac, C.; Witulski, B.; Hofmann, T. Structure determination of

501

bisacetylenic oxylipins in carrots (Daucus carota L.) and enantioselective

502

synthesis of falcarindiol. J. Agric. Food Chem. 2009, 57, 11030–11040.

503

(19) Schmiech, L. Strukturaufklärung und quantitative Studien zu Bitterstoffen und

504

Polyacetylenen in Karotten (Daucus carota L.). Technische Universität

505

München. Thesis. 2010. ACS Paragon Plus Environment

20

Page 21 of 41

506 507


(20) Bohlmann, F.; Zdero, C. A novel polyyne from Centella species (in German). Chem. Ber. 1975, 108, 511–514.

508

(21) Fujioka, T.; Furumi, K.; Fujii, H.; Okabe, H.; Muhashi, K.; Nakano, Y.;

509

Matsunaga, H.; Katano, M.; Mori, M. Anti-proliferative constituents from

510

umbelliferae plants. V. A new furanocoumarin and falcarindiol furanocoumarin

511

ethers from the root of Angelica japonica. Chem. Pharm. Bull. 1999, 47, 96–

512

100.

513

(22) Kobaisy, M.; Abramowski, Z.; Lermer, L.; Saxena, G.; Hancock, R. E. W.;

514

Towers, G. H. N. Antimycobacterial polyynes of Devil Club (Oplopanax

515

horridus), a North American native medical plant. J. Nat. Prod. 1997, 60, 1210–

516

1213.

517 518

(23) Shim, S. C.; Chang, S.-K.; Hur, C. W.; Kim, C. K. A polyacetylenic compound from Panax ginseng roots. Phytochemistry 1987, 26, 2849–2850.

519

(24) Zidorn, C.; Jöhrer, K.; Ganzera, M.; Schubert, B.; Sigmund, E. M.; Mader, J.;

520

Greil, R.; Ellmerer, E. P.; Stuppner, H. Polyacetylenes from the Apiaceae

521

vegetables carrot, celery, fennel, parsley and parsnip and their cytotoxic

522

activities. J. Agric. Food Chem. 2005, 53, 2518–2523.

523

(25) Larsen, P. K.; Nielsen, B. E.; Lemmich, J. Constituents of umbelliferous plants

524

XII. The absolute configuration of falcarinol, and acetylene rich compound from

525

roots of Seseli gummiferum Pall. Acta Chem. Scand. 1969, 23, 2552–2554.

526

(26) Lemmich, E. The absolute configuration of the acetylenic compound falcarindiol.

527

Phytochemistry 1981, 20, 1419–1420.

528

(27) Bernart, M. W.; Cardellina, J. H.; Balaschak, M.; Alexander, M.; Shoemaker, R.;

529

Boyd, M. Cytotoxic falcarinol oxylipins from Dendropanax arboreus. J. Nat.

530

Prod. 1996, 59, 748–753.


21


Page 22 of 41

531

(28) Zheng, G.; Lu, W.; Cai, J. Stereoselective total synthesis of (3R,8S)-falcarindiol,

532

a common polyacetylene compound from Umbellifers. J. Nat. Prod. 1999, 62,

533

626–628.

534 535

(29) Shun, S.; Tykwinski, R. R. Synthesis of naturally occurring polyenes. Angew. Chem., Int. Ed. 2006, 45, 1034–1057.

536

(30) Sabitha, G.; Bhaskar, V.; Reddy, Ch.; Yadav, J. S. Stereoselective approaches

537

for the total synthesis of polyacetylenic (3R,8S)-falcarindiol. Synthesis. 2008, 1,

538

115–121.

539

(31) Rai, D. K.; Brunton, N. P.; Koidis, A.; Rawson, A.; McLoughlin, P.; Griffiths, W.

540

J. Characterisation of polyacetylenes isolated from carrot (Daucus carota)

541

extract by negative ion tandem mass spectrometry. Rapid Commun. Mass

542

Spectrom. 2011, 25, 2231–2239.

543

(32) Kidmose, U.; Hansen, S.L.; Christensen, L.P.; Edelenbos, M.; Larsen, E.;

544

Nørbæk, R. Effects of genotype, root size, storage, and processing on bioactive

545

compounds in organically grown carrots (Daucus carota L.). J. Food Sci. 2004,

546

69, S388−S394.

547

(33) Zidorn, C.; Johrer, K.; Ganzera, M., Schubert, B.; Sigmund, E. M.; Mader, J.;

548

Greil, R.; Ellmer, E. P.; Stuppner, H. Polyacetylenes from the Apiaceae

549

vegetables carrot, celery, fennel, parsley, and parsnip and their cytotoxic

550

activities. J. Agric. Food Chem. 2005, 53, 2518–2523.

551

(34) Czepa, A.; Hofmann, T. Quantitative studies and sensory analyses on the

552

influence of cultivar, spatial tissue distribution, and industrial processing on the

553

bitter off-taste of carrots (Daucus carota L.) and carrot products. J. Agric. Food

554

Chem. 2004, 52, 4508–4514.


22

Page 23 of 41


555

(35) Nitz, S.; Spraul, M. H.; Drawert, F. C17 Polyacetylenic alcohols as the major

556

constituents in roots of Petroselinum crispum Mill. ssp. tuberosum. J. Agric.

557

Food Chem. 1990, 38, 1445–1447.

558

(36) Santos, P. A. G.; Figueiredo, A. C.; Oliveira, M. M.; Barroso, J. G.; Pedro, L. G.;

559

Deans, S. G.; Scheffer, J. J. C. Growth and essential oil composition of hairy

560

root cultures of Levisticum officinale W.D.J. Koch (lovage). Plant Sci. 2005, 168,

561

1089–1096.

562

(37) Brandt, K.; Christensen, L. P.; Hansen-Møller, J.; Hansen, S. L.; Haraldsdóttir,

563

J.; Jespersen, L.; Purup, S.; Kharazmi, A.; Barkholt, V.; Frøkiær, H.; Kobæk-

564

Larson, M. Health promoting compounds in vegetables and fruits: A systematic

565

approach for identifying plant components with impact on human health. Trends

566

Food Sci. Technol. 2004, 15, 384–393.

567 568

(38) Schulz, H.; Baranska, M. Identification and quantification of valuable plant substances by IR and Raman spectroscopy. Vib. Spectrosc. 2007, 43, 13–25.

569

(39) Killeen, D. P.; Sansom, C. E.; Lill, R. E.; Eason, J. R.; Gordon, K. C.; Perry, N.

570

B. Quantitative raman spectroscopy for the analysis of carrot bioactives. J.

571

Agric. Food Chem. 2013, 61, 2701−2708.

572

(40) Baranska, M.; Schulz, H.; Baranski, R.; Nothnagel, T., Christensen, L. P. In situ

573

simultaneous analysis of polyacetylenes, carotinoids and polysaccharides in

574

carrot roots. J. Agric. Food Chem. 2005, 53, 6565–6571.

575

(41) Harding, V. K.; Heale, J. B. The accumulation of inhibitory compounds in the

576

induced resistance response of carrot root slices to Botrytis cinerea.

577

Physiological Plant Pathology 1981, 18(1), 7–15.

578

(42) Olsson, K. and Svensson, R. The influence of polyacetylenes on the

579

susceptibility of carrots to storage diseases. Journal of Phytopathology-

580

Phytopathologische Zeitschrift 1996, 144(9-10), 441–447. ACS Paragon Plus Environment

23


581 582

Page 24 of 41

(43) Lund, E. D.; White, J. M. Polyacetylenes in Normal and waterstressed 'Orlando Gold' carrots (Daucus carota). J. Sci. Food Agric. 1990, 51, 507–516.

583

(44) Kreutzmann, S.; Christensen, L. P.; Edelenbos, M. Investigation of bitterness in

584

carrots (Daucus carota L.) based on quantitative chemical and sensory

585

analyses. LWT - Food Science and Technology (2008 a), 41(2), 193–205.

586

(45) Kreutzmann, S.; Svensson, V. T.; Thybo, A. K.; Bro, R.; Petersen, M. A.

587

Prediction of sensory quality in raw carrots (Daucus carota L.) using multi-block

588

LS-ParPLS. Food Qual. Pref. 2008, 19, 609–617.

589 590

(46) Lund, E. D.; Bruemmer, J. H. Acetylenic compounds in stored packaged carrots. J. Sci. Food Agric. 1991, 54, 287–294.

591

(47) Lund, E. D.; Marion, W. J. Polyacetylenes in normal and water stressed

592

´Orlando Gold´ carrots (Daucus carota L.). J. Sci. Food Agric. 1990, 51, 507–

593

516.

594

(48) Pferschy-Wenzig, E. M.; Getzinger, V.; Kunert, O.; Woelkart, K.; Zahrl, J.;

595

Bauer, R. Determination of falcarinol in carrot (Daucus carota L.) genotypes

596

using liquid chromatography/mass spectrometry. Food Chem. 2009, 114(3),

597

1083–1090.

598 599 600

(49) Schulz-Witte, J. Diversität wertgebender Inhaltsstoffe bei Daucus carota L. Thesis, Technical Univ. Braunschweig, 2011. (50) Schulz-Witte, J.; Nothnagel, T.; Schulz, H. Comparison of different clean-up

601

methods

602

polyacetylenes in carrot roots. J Appl. Botan. Food Qual. 2010, 83, 123–127.

603 604 605 606

for

simultaneous

HPLC

determination

of

carotenoids

and

(51) Baranska, M.; Schulz, H. Spatial tissue distribution of polyacetylenes in carrot root. Analyt 2005, 130, 855–859. (52) Minto, R. E.; Blacklock, B. J. Biosynthesis and function of polyacetylenes and allied natural products. Prog. Lipid Res. 2008, 47, 233–306. ACS Paragon Plus Environment

24

Page 25 of 41


607

(53) Lecomte, M.; Berruyer, R.; Hamamaa, L.; Boedo, C.; Hudhommed, P.;

608

Bersihand, S.; Arul, J.; N´Guyen, G.; Gatto, J.; Guilet, D.; Richomme, P.;

609

Simoneau, P.; Briad, M.; Le Clerc, V.; Poupard, P. Inhibitory effect of the carrot

610

metabolites 6-methoxymellein and falcarindiol on development of the fungal leaf

611

blight pathogen Alternaria dauci. Phys. Mol. Plant Path. 2012, 80, 58–67.

612 613 614 615

(54) Rubatzky, V. E., Quiros, C. F., Simon, P. W. Carrots and related vegetable Umbelliferae. CABI Publishing, New York, 1999. (55) Alessandro, M. S. and Galmarini, C. R. Inheritance of vernalization requirement in carrot. J. Amer. Soc. Hort. Sci. 2007,132, 525-529.

616

(56) Alessandro, M. S., Galmarini, C. R., Iorizzo, M., and Simon, P. W. (2013)

617

Molecular mapping of vernalization requirement and fertility restoration genes in

618

carrot. Theor. Appl. Genet. 2013, 126, 415-423.

619

(57) Schieberle, P.; Hofmann, T. Mapping the combinatorial code of food flavours by

620

means of molecular sensory science approach. In: Food Flavors – Chemical,

621

Sensory and Technological Properties (Jelen, H.; Ed.); CRC Press, Taylor and

622

Francis Group 2011, 411–437.

623

(58) Kreutzmann, S.; Christensen, L. P.; Edelenbros, M. Investigation of bitterness in

624


625

analyses. LWT - Food Sci. Technol. 2008, 41, 193–205.

626 627 628

(59) Yamaguchi, M.; Howard, F. D.; McNelly, L. B. Bitterness of California-grown carrots. Plant Dis. Rep. 1955, 39, 302–304. (60) Sondheimer, E. Bitter flavor in carrots. IV. The isolation and identification of 3-

629

methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin

630

Chem. Soc. 1957, 79, 5036–9.

from

carrots.

J.

Am.

631

(61) Seljåsen, R.; Bengtsson, G. B.; Hoftun, H.; Vogt, G. Sensory and chemical

632

changes in five varieties of carrot (Daucus carota L.) in response to mechanical

633

stress at harvest and post-harvest. J. Sci. Food Agric. 2001, 81, 436–447. ACS Paragon Plus Environment

25


Page 26 of 41

634

(62) Seljåsen, R.; Hoftun, H.; Bengtsson, G. B. Sensory quality of ethylene-exposed

635

carrots (Daucus carota L, cv ”Yukon”) related to the contents of 6-

636

methoxymellein, terpenes and sugars. J. Sci. Food Agric. 2001, 81, 54–61.

637

(63) Seljåsen, R.; Vogt, G.; Olsen, E.; Lea, P.; Høgetveit, L. A.; Tajet, T. Influence of

638

field attack by carrot Psyllid (Trioza apicalis Förster) on sensory quality,

639

antioxidant capacity and content of terpenes, falcarindiol and 6‑methoxymellein

640

of carrots (Daucus carota L.). J. Agric. Food Chem. 2013, 61, 2831−2838.

641

(64) Kreutzmann, S.; Christensen, L. P.; Edelenbos, M. Investigation of bitterness in

642


643

analyses. Lebensm. Wiss. Technol. 2008, 41, 193–205.

644

(65) Cao, S.; Zhou, X.-R.; Wood, G. C.; Green, A. G.; Singh, S. P.; Liu, L.; Liu, Q. A

645

large and functionally diverse family of Fad2 genes in safflower (Carthamus

646

tinctorius L.). BMC Plant Biology 2013, 13, 5.

647

(66) Barley, G. C.; Jones, H. E. R.; Thaller, V. Crepenynate as a precursor of

648

falcarinol in carrot tissue culture. In: Chemistry and Biology of Naturally-

649

Occurring Acetylenes and Related Compounds; Lam, J. et al. Eds.; Elsevier:

650

Amsterdam, The Netherlands, 1988, 85–91.

651

(67) Knispel, N.; Ostrozhenkova, E.; Schramek, N.; Huber, C.; Peña-Rodríguez L.

652

M., Bonfill, M.; Palazón, J.; Wischmann, G.; Cusidó, R. M.; Eisenreich, W.

653

Biosynthesis of Panaxynol and Panaxydol in Panax ginseng. Molecules 2013,

654

18, 7686–7698.

655

(68) Okuley, J.; Lightner, J.; Feldmann, K.; Yadav, N.; Lark, E.; Browse, J.

656

Arabidopsis

657

polyunsaturated lipid synthesis. Plant Cell 1994, 6, 147–158.

658 659

FAD2

gene

encodes

the

enzyme

that

is

essential

for

(69) Shanklin, J.; Cahoon, E. B. Desaturation and related modifications of fatty acids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998, 49, 611–641. ACS Paragon Plus Environment

26

Page 27 of 41


660

(70) Van Der Loo, F. J.; Broun, P.; Turner, S.; Somerville, C. An oleate 12-

661

hydroxylase from Ricinus communis L. is a fatty acyldesaturase homolog. Proc.

662

Natl. Acad. Sci. USA 1995, 92, 6743–6747.

663

(71) Broun, P.; Somerville, C. Accumulation of ricinoleic, lesquerolic, and densipolic

664

acids in seeds of transgenic arabidopsis plants that express a fatty acyl

665

hydroxylase cDNA from castor bean. Plant Physiol. 1997, 113, 933–942.

666

(72) Lee, M.; Lenman, M.; Banaś, A.; Bafor, M.; Singh, S. P.; Schweizer, M.;

667

Nilsson, R.; Liljenberg, C.; Dahlqvist, A.; Gummeson, P. O.; Sjödahl, S.; Green,

668

A.; Stymne, S. Identification of non-heme diiron proteins that catalyze triple

669

bond and epoxy group formation. Science 1998, 280, 915–918.

670

(73) Cahoon, E. B.; Kinney, A. J. Dimorphecolic acid is synthesized by the

671

coordinate activities of two divergent delta12-oleic acid desaturases. J. Biol.

672

Chem. 2004, 279, 12495–12502.

673 674

(74) Broun, P.; Boddupalli, S.; Somerville, C. A bifunctional oleate 12-hydroxylase desaturase from Lesquerella fendleri. Plant J. 1998, 13, 201–210.

675

(75) Carlsson, A. S.; Thomaeus, S., Hamberg, M.; Stymne, S. Properties of two

676

multifunctional plant fatty acid acetylenase/desaturase enzymes. Eur. J.

677

Biochem. 2004, 271, 2991–2997.

678

(76) Heppard, E. P.; Kinney, A. J.; Stecca, K. L.; Miao, G. H. Developmental and

679

growth temperature

regulation

of

two different

microsomal

680

desaturase genes in soybeans. Plant Physiol. 1996, 110, 311–319.

[omega]-6

681

(77) Scheffler, J. A.; Sharpe, A. G.; Schmidt, H.; Sperling, P.; Parkin, I. A.; Lühs, W.;

682

Lydiate, D. J.; Heinz, E. Desaturase multigene families of Brassica napus arose

683

through genome duplication. Theor. Appl. Genet. 1997, 94, 583–591.

684

(78) Liu, Q.; Singh, S. P.; Brubaker, C. L.; Sharp, P. J; Green, A. G.; Marshall, D. R.

685

Molecular cloning and expression of a cDNA encoding a microsomal ω-6 fatty ACS Paragon Plus Environment

27


Page 28 of 41

686

acid desaturase from cotton (Gossypium hirsutum). Funct. Plant Biol. 1999, 26,

687

101–106.

688

(79) Jung, S.; Swift, D.; Sengoku, E.; Patel, M.; Teulé, F.; Powell, G., Moore, K.;

689

Abbott, A. The high oleate trait in the cultivated peanut [Arachis hypogaea L.]. I.

690

Isolation and characterization of two genes encoding microsomal oleol-PC

691

desaturases. Mol. Gen. Genet. 2000, 263, 796–805.

692

(80) Khadake, R. M.; Ranjekar, P. K.; Harsulkar, A. M. Cloning of a novel omega-6

693

desaturase from flax (Linum usitatissimum L.) and its functional analysis in

694

Saccharomyces cerevisiae. Mol. Biotechnol. 2009, 42, 168–174.

695

(81) Martínez-Rivas, J. M.; Sperling, P.; Lühs, W.; Heinz, E. Spatial and temporal

696

regulation of three different microsomal oleate desaturase genes (FAD2) from

697

normal-type and high-oleic varieties of sunflower (Helianthus annuus L.). Mol.

698

Breed. 2001, 8, 159–168.

699

(82) Qiu, X.; Reed, D. W; Hong, H.; MacKenzie, S. L; Covello, P. S. Identification

700

and analysis of a gene from Calendula officinalis encoding a fatty acid

701

conjugase. Plant Physiol. 2001, 125, 847–855.

702

(83) Cahoon, E. B.; Schnurr, J. A.; Huffman, E. A.; Minto, R. E. Fungal responsive

703

fatty acid acetylenases occur widely in evolutionarily distant plant families. Plant

704

J. 2003, 34, 671–683.

705

(84) Li, C.; Zhu, Y.; Guo, X.; Sun, C.; Luo, H., Song, J.; Li, Y.; Wang, L.; Qian, J.;

706

Chen, S. Transcriptome analysis reveals ginsenosides biosynthetic genes,

707

microRNAs and simple sequence repeats in Panax ginseng C. A. Meyer. BMC

708

Genomics 2013, 14, 245.

709

(85) Liu, R. H. Health benefits of fruit and vegetables are from additive and

710

synergistic combinations of phytochemicals. Am. J. Clin. Nutr. 2003, 78, 517–

711

520. ACS Paragon Plus Environment

28

Page 29 of 41


712

(86) Crozier, A., Jaganath, I. B.; Clifford, M. N. Dietaryl phenolics: chemistry,

713

bioavailability and effects on health. Nat. Prod. Rep. 2009, 26, 1001–1043.

714

(87) Tan, K. W.; Killeen, D. P.; Li, Y.; Paxton, J. W.; Birch, N. P.; Scheepens, A.

715

Dietary polyacetylenes of the falcarinol type are inhibitors of breast cancer

716

resistance protein (BRCP/ABCG2). Eur. J. Pharmacology 2013, 723, 346–352.

717

(88) Kobæk-Larsen, M.; Christensen, L. P.; Vach, W., Ritskes-Hoitinga, J.; Brandt,

718

K. Inhibitory effects of feeding with carrots or (-)-falcarinol on development of

719

azoxymethane-induced preneoplastic lesions in the rat colon. J. Agric. Food

720

Chem. 2005, 53, 1823–1827.

721 722

(89) Ahn, B.-Z.; Kim, S.-I. Relation between structure and cytotoxic activity of panaxydol analogues against L1210 cells. Arch. Pharm. 1988, 321(2), 61–63.

723

(90) Matsunaga, H.; Katano, M.; Yamamoto, H.; Mori, M.; Takata, K. Cytotoxic

724

activity of polyacetylene compounds in Panax ginseng C. A. Meyer. Chem.

725

Pharm. Bull. 1990, 38(12), 3480–3482.

726

(91) Matsunaga, H., Katano, M.; Yamamoto, H.; Fujito, H.; Mori, M.; Takata, K.

727

Studies on the panaxytriol of Panax ginseng C. A. Meyer. Isolation,

728

determination and antitumor activity. Chem. Pharm. Bull. 1989, 37(5), 1279–

729

1281.

730

(92) Hansen, S. L.; Purup, S.; Christensen, L. P. Bioactivity of falcarinol and the

731

influence of processing and storage on its content in carrots (Daucus carota L.).

732

J. Sci. Food Agric. 2003, 83, 1010–1017.

733 734

ABBREVIATIONS ABCG2

The second member of the G subgroup of human ABC transporter proteins, also known as BCRP


29


A. dauci

Alternaria dauci

Batt.

Jules Aimé Battandier

BCRP/ABCG2

Breast cancer resistance protein, ABCG2

BLAST

Basic Local Alignment Search Tool

C. alpina

Crepis alpina L.

D. c.

Daucus carota L.

D. carota

Daucus carota L.

Desf.

René Louiche Desfontaines

DoT

Dose-over-threshold

EST

expressed sequence tag

FAD2

∆12-fatty acid desaturase

FaDOH

falcarindiol

FaDOH3Ac

falcarindiol-3-acetate

FaOH

falcarinol

H. annuus

Helianthus annuus

Hoffm.

Georg Franz Hoffmann

HPLC

High-performance liquid chromatography

L.

Carl von Linné

Lam.

Jean-Baptiste Lamarck

MAS

marker assisted selection

Merr.

Elmer Drew Merrill

Mbp

Mega base pairs

Mill.

Philip Miller

MlQ.

Friedrich Anton Wilhelm Miquel

NGS

next generation sequencing


Page 30 of 41

30

Page 31 of 41


PA

polyacetylene

(RT)-PCR

Reverse transcription polymerase chain reaction

SM.

James Edward Smith

ssp.

subspecies

735 736

ACKNOWLEGEMENT

737

The authors thank Dr. R. Lang for his critical reading of the manuscript.

738 739


31


740

Page 32 of 41

FIGURE LEGEND

741 Figure 1.

Chemical structures of bisacetylenes identified in carrots: falcarinol (1), falcarindiol

(2),

falcarindiol-3-acetate

falcarindiol

8-acetate

falcarindiolone-8-acetate

(5),

(3),

(E)-isofalcarinolone

1,2-dihydrofalcarindiol-3-acetate

(7),

(E)-falcarindiolone-9-acetate

(4),

(6),

(E)-

(8),

1,2-

dihydrofalcarindiol (9), (E)-1-methoxy-falcarindiolone-8-acetate (10), (E)-1methoxy-falcarindiolone-9-acetate (11), and panaxydiol (12). Figure 2.

Enantioselective synthesis of the (3R,8S), (3S,8S), (3S, 8R), and (3R, 8R) diastereomers of falcarindiol (adapted by Schmiech et al.18).

Figure 3.

Scatterplots of individual plant analyses of the polyacetylenes falcarinol, falcarindiol and falcarindiol-3-acetate (plants grown as single plant pot culture under optimized greenhouse conditions in frame of an association study, red - data of 303 individual plants from 100 carrot cultivars, blue – 286 individuals from 100 Daucus wild relatives, mg/100g FW (taken from Schulz-Witte49).

Figure 4.

Pictures of carrots (a-c) and wild (d-f) carrots cultivated in a sand-humus mixture (3:1) in plastic pots (∅19cm/H27cm) and under optimized glasshouse conditions (i.e. 20-25oC/10-15oC D/N-temperature, ~ 60% rH, drop irrigation): cv. Nevis (F1, Bejo, NL) (a), cv. Rotin (OP, Sperling, Germany) (b), Landrace fromArmenia (VIR, St. Petersburg, RU) (c), D. c. ssp. carota (Germany) (d), D. c. ssp. carota (Italy) (e), and D.c. ssp. carota (United Kingdom) (f). Size marker (white label) 14 x 2 cm.

Figure 5.

Possible biosynthetic pathway of falcarinol-type C17-polyacetylenes in higher plants like carrots (adapted by Minto and Blacklock52).


32

Page 33 of 41


Figure 6.

(A) Phylogenetic tree of the deduced Daucus FAD2 proteins (numbered from 1 to 6), a published partial Daucus acetylenase (DcACET), the published Petroselinum ELI12 acetylenase, a Panax ginseng FAD2 and some other putative acetylenases and desaturases / hydroxylases (numbers are

NCBI accession numbers; Dc, Daucus carota; Pc,

Petroselinum crispum; Pg, Panax ginseng; Ca, Crepis alpina; Ha, Helianthus annuus; At, Arabidopsis thaliana; Bo, Borago officinalis; Cp, Crepis palaestina; Ah, Arachis hypogaea). (B) Multiple sequence alignment of the deduced proteins shown in Figure 6A. Only the amino acid positions 101 – 150 are shown which include the first histidine motif HEC(G/D)H at aa positions 109-113 and the G/A residue

at

position

108

possibly

indicating

acetylenase

activity

(abbreviations see Fig. 6A). 742 743 744 745 746 747 748 749 750 751 752 753 754 ACS Paragon Plus Environment

33


Page 34 of 41

755

Table 1. Contents of Falcarinol-type PAs in Carrot Cultivars and Wild Daucus Relatives

756

(FaOH, Falcarinol; FaDOH, Falcarindiol; FaDOH3Ac, Falcarindiol-3-acetate) according to

757

Schulz-Witte49

758 PA content (mg / 100 mg FW) D. carota ssp. sativus

Geographic

FaOH

FaDOH

FaDOH3Ac

Total

USA

28.1

42.4

14.9

85,4

cv. Yellowstone

Netherlands

6.2

34.9

6.8

47.8

cv. Deep Purple (F1)

Netherlands

16.2

16.8

2.5

35.5

cv. Regulus Imperial

Sweden

2.0

27.6

5.1

34.8

cv. Blanche long des vosges

France

6.2

20.0

8.2

34.4

cv. Schweizer Rübli

Germany

2.3

21.4

8.6

32.4

cv. Nantes Empire

France

4.6

23.5

3.7

31.8

Pakistan

10.0

19.9

1.4

31.4

cv. Vita Longa

Netherlands

1.0

23.0

2.2

26.2

cv. White Satin (F1)

Netherlands

2.8

18.6

4.5

26.0

Germany

0.4

3.3

0.2

3.9

Netherlands

0.8

2.1

0.5

3.4

France

0.5

2.5

0.4

3.3

D. c. ssp. maritimus

Spain

31.8

465.2

40,0

537.0

D. c. ssp. halophilus

Portugal

13.0

366.6

3.9

383.5

D. c. ssp. azoricus

Azores

21.4

202.6

21.0

245.1

Italy

87.2

132.4

14.9

234.5

D. c. ssp. gummifer

France

60.2

152.7

15.7

228.6

D. c. ssp. maximus

Portugal

11.6

201.5

8.9

222.0

D. c. ssp. carota

Europe

107.3

100.3

5.3

212.9

D. c. ssp. gadecai

Spain

54.2

133.5

12.8

200.5

D. c. ssp. gummifer

France

30.1

155.6

14.2

200.0

D. c. ssp. maritimus

Spain

49.1

134.2

7.8

191.1

D. c. ssp. major

France

25.5

138.9

9.1

173.6

D. c. ssp. gummifer

Spain

14.9

138.9

10.3

164.1

D. c. ssp. carota

France

22.3

131.2

7.8

161.2

D. c. ssp. carota

Greece

11.9

110.5

4.8

127.1

D. c. ssp. carota

Germany

12.7

97.6

15.5

125.8

cv. Anthonina

cv. Gajar

cv. Lange Rote Stumpfe cv. Purple Haze (F1) cv. Presto

origin

D. carota ssp.

D. c. ssp. commutatus

759 760


34

Page 35 of 41


Dawid et al., Figure 1


35


Page 36 of 41


H2/Pd/KOH/chinoline

Ph Ph

C6H13

H

TMS

S

S/R

HN

C6H13

TMS

B

O TMS

PhI(OAc)2/ TEMPO(cat.)

(E)/(Z): 15/85 99%

CBS reduction with Garcia ligand

H Ph Ph

HN(CH3)-OCH3*HCl

O

TMS N

pyrid., CH2Cl2, 0°C

O

60%

R/S

c/d

+

O

TMS

S/R

a/b

C6H13

O

HO

B

H TMS

R

c

TMS

THF, 0°C

OH

H TMS

MgBr

BH3*SMe2, THF, 0°C

60%

H R/S

TMS (E)/(Z): 12/88 98%

HN

HO

H

HO C6H13

CH2Cl2

Ph

OH

BuLi TMS

O

b

O

O (E)/(Z): 7/93

THF -78°C

BH3*SMe2, THF, 0°C

R

C6H13

CH2Cl2

C6H13

OH

PhI(OAc)2/ TEMPO(cat.)

Ph

a H

OH

EtOH

OH

HO H

C6H13

H

CBS70% reduction with Garcia ligand

CuCl, NH2OH*HCl, C4H9NH2 MeOH, 0°C

OH TMS

S

d

HO

OH R/S

S/R

C6H13

25%


(3R,8S), (3S,8R), (3R,8R), (3S,8S)

36

Page 37 of 41



37 ACS Paragon Plus Environment


Page 38 of 41



38

Page 39 of 41


Figure 5


39


Page 40 of 41



40

Page 41 of 41


TOC Graphic 235x94mm (150 x 150 DPI)


Bioactive C17-Polyacetylenes in Carrots (Daucus ... - ACS Publications

Recommend Documents