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

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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

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Journal of Agricultural and Food Chemistry

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

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Current Knowledge and Future Perspectives

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

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Nothnagel§, and Thomas Hofmannǂ*

6 ǂ

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Chair for Food Chemistry and Molecular Sensory Science, Technische Universität

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

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

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

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Quedlinburg, Germany #

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Biotechnology of Natural Products, Technische Universität München, Liesel-

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Beckmann-Strasse 1, D-85354 Freising, Germany

14 ≠

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These authors contributed equally to this work.

16 17

Running Title: Bioactive polyacetylenes in carrots.

18 19

*

20

PHONE

+49-8161/71-2902

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FAX

+49-8161/71-2949

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E-MAIL

[email protected]

Author to whom correspondence should be addressed

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ACS Paragon Plus Environment

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ABSTRACT

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

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

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

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

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serotogenic effects. These findings suggest targeting vegetables with elevated levels

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

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

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

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

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

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to improving human health and well-being.

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

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bitterness, Daucus carota L.

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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inflammatory properties at nontoxic concentrations for humans.1-3

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

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

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

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

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

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

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

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future knowledge-based carrot breeding programs aimed at elevating the

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concentration of polyacetylenes in specific carrot cultivars holding potential to

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contribute to human health management.

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CARROTS AND THEIR POLYACETYLENES

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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functional gene studies.16 ACS Paragon Plus Environment

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Bisacetylenic Oxylipins in Carrots. Although first phytochemicals have been

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

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

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has been focused on the polyacetylenes´ chemical structures and biological

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

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subset of 12 structurally related bisacetylenic oxylipins were isolated from Daucus

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

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

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

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

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

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

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

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these quantitatively predominating polyacetylenes, nine additional bisacetylenes

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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PAs

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

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

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easily be assigned by comparing 1H and

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revealed typical chemical

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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Quantification and Distribution of Polyacetylenes in Daucus carota. An

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

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or food supplements is the reliable molecular characterization of the bioactive

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

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

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chromatographic isolation of reference materials needed to study their bioactivity in

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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comparison, the cultivars contained explicitly lower concentrations of PAs ranging

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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.

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

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

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

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

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

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the contents of each individual PA separately.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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accumulation of polyacetylenes like falcarinol was observed in tomato fruits and

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

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Berth., and Fusarium oxysporum Schlecht.52

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

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

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

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

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

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as UV irradiation or salt stress.52

In carrots, the development of the

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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an economically drug production on a field-scale basis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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considerations on key phytochemicals of carrots influenced by those different

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external factors are available.

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BIOSYNTHESIS

AND

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POLYACETYLENES

MOLECULAR

GENETICS


OF

CARROT

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Despite the extensive research concerning the analytical and biochemical

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

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

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

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

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

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

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concentrations of specific PAs in (crop) plants.

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

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

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

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Solanaceae.52 The crepenynate pathway for acetylenic natural product biosynthesis

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

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

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

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

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

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

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molecules such as falcarinol. In D. carota,

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

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

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

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CoA via crepenynate as the putative intermediate.67

14

C-labeled crepenynic acid has been

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

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

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

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

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

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

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

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

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

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

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

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

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

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fatty acid are designated as acetylenases.75

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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

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results in a

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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

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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

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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

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(1)

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

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

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2010, 285–306.

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

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

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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

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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


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38

Page 39 of 41


Figure 5


39


Page 40 of 41



40

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TOC Graphic 235x94mm (150 x 150 DPI)


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

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