Comparative Analysis of Two Flavonol Synthases from Different

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Comparative analysis of two flavonol synthases from differentcolored onions provides insight into flavonoid biosynthesis Sangkyu Park, Da-Hye Kim, Jong-Yeol Lee, Sun-Hwa Ha, and Sun-Hyung Lim J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

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Journal of Agricultural and Food Chemistry

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Comparative analysis of two flavonol synthases from different-colored onions provides

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insight into flavonoid biosynthesis

3 4 5 6

Sangkyu Park,† Da-Hye Kim,† Jong-Yeol Lee,† Sun-Hwa Ha,§ and Sun-Hyung Lim*,† †

National Institute of Agricultural Science, Rural Development Administration, JeonJu,

7

54874, Republic of Korea; [email protected] (S.P.); [email protected] (D.H.K.);

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[email protected] (J.Y.L.)

9

§

Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee

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University, Yongin, 17104, Republic of Korea; [email protected]

11

*

Correspondence: [email protected]; Tel.: +82-63-238-4615

12 13 14 15 16 17 18 19 20 21 22 ACS Paragon Plus Environment


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Abstract

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We isolated cDNAs encoding flavonol synthase (FLS) from the red onion ‘H6’ (AcFLS-H6)

25

and the yellow onion ‘Hwangryongball’ (AcFLS-HRB). We found three amino acid

26

variations between the two sequences. Kinetic analysis with recombinant proteins revealed

27

that AcFLS-HRB exhibited approximately 2-fold higher catalytic efficiencies than

28

AcFLS-H6 for dihydroflavonol substrates, and both proteins preferred dihydroquercetin to

29

dihydrokaempferol. The expression patterns of flavonoid biosynthesis genes corresponded to

30

the accumulation patterns of flavonoid aglycones in both onions. Whereas the other

31

flavonoid biosynthesis genes were weakly expressed in HRB sheath compared to that of H6,

32

the expression of FLS was similar in both onions. This relatively enhanced FLS expression,

33

along with the higher activity of AcFLS-HRB, could increase the quercetin production in

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HRB sheath. The quercetin content was approximately 12-fold higher than the cyanidin

35

content in H6 sheath, suggesting that FLS has priority in the competition between FLS and

36

DFR for their substrate dihydroquercetin.

37 38 39

Keywords

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anthocyanins; flavonoid biosynthetic pathway; flavonol; flavonol synthase; kaempferol;

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onion; quercetin

42 43 44 ACS Paragon Plus Environment

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Introduction

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Flavonoids are polyphenolic secondary metabolites naturally found in the plant kingdom.

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To date, approximately 10,000 different flavonoids have been identified, 1 and their wide

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array of physiological roles in plants2 and nutritional and pharmacological benefits in terms

49

of human health3 have been elucidated. The structure of flavonoids includes a 15-carbon

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phenylpropanoid core, which can be modified by rearrangement, alkylation, oxidation, and

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glycosylation.4 Flavonoids can be divided into various subclasses: flavones, isoflavones,

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dihydroflavonols, flavonols, flavan-3-ols, and anthocyanins, depending on the modification

53

of the C-ring.5 Among these subclasses, flavonols are the most prevalent in plants and are

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mainly represented by glycosides of kaempferol, quercetin, myricetin, and isorhamnetin.

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Flavonols play crucial roles in plants including in protection against UV-B irradiation6 and

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pathogenic microorganisms,7 regulation of auxin transport,8 signaling for symbiont

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attraction,9 and male fertility.10,11 Besides their physiological functions in plants, diverse

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health-promoting properties, such as antioxidant, anti-proliferative, anti-angiogenic, and

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neuropharmacological effects, have also been demonstrated.12,13

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The formation of flavonol is catalyzed by the action of flavonol synthase (FLS) (EC:

61

1.14.11.23) (Figure 1). FLS activity was first observed in protein extracts from irradiated

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parsley

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2-oxoglutarate-dependent dioxygenase (2-ODD), a non-heme ferrous-containing cytosolic

64

enzyme that requires 2-oxoglutarate as a co-substrate.14 FLS cDNAs have been characterized

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from various plants including Arabidopsis thaliana,15 Camellia sinensis,16 Citrus unshiu,17

66

Fagopyrum tataricum,18 Ginkgo biloba,19 Petunia hybrida,20 Vitis vinifera,21 and Zea mays.22

(Petroselinum

hortense)

cells

and

characterized

ACS Paragon Plus Environment

in

vitro

as

a


67

In addition to FLS, flavanone 3-hydroxylase (F3H), flavone synthase I (FNSI), and

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anthocyanidin synthase (ANS) also belong to a family of 2-ODDs in the flavonoid

69

biosynthetic pathway.4 The cDNA sequences encoding FLSs from various species share

70

significant levels of similarity (50%-60%) with those encoding ANS, but much less

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similarity with those encoding FNSI or F3H.23

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Onions are one of the richest sources of dietary flavonoids and the second-most produced

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vegetable crop after tomatoes.24,25 Although the quality and quantity of the flavonoid pool

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in onions are highly variable depending on the cultivar, growth stage, environmental

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condition, and post-harvest practice, the main flavonoids in onions are flavonols.26 At least

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25 different flavonols have been characterized from onion bulbs, among which quercetin

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derivatives are the most prevalent.27 Specifically, quercetin 4'-glucoside and quercetin

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3,4'-diglucoside are the main flavonols in onion bulbs, accounting for approximately 80-95%

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of all flavonoids in white, yellow, and red onions.26-34 Additionally, red onions contain

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substantial levels of anthocyanins, mainly cyanidin derivatives such as cyanidin 3-glucoside

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and cyanidin 3-(6''-malonylglucoside), but their levels are only approximately 10% of the

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total flavonoids.26 Glycosylation of the onion flavonoids and the subsequent malonylation

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on the glycosyl moiety improve the solubility and stability of the flavonoid structures.35

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The glucose-conjugated flavonoids in onions are known to be more efficient for internal

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absorption compared to pure aglycones or the flavonoids conjugated with galactoside,

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rhamnoside, arabinoside, or xyloside that are found in other flavonoid-containing fruits and

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vegetables.26,32 The importance of onions in the human diet has led to a number of efforts to


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maximize the biosynthesis of specific flavonoids in onions 26,31,36 and to identify the related

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genes.

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Nearly all of the structural genes involved in the flavonoid pathway have been identified

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in many different species.37 In the case of onions, the pathway is inferred as shown in

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Figure 1 on the basis of the facts that quercetin derivatives are the most abundant flavonoid

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in onions and anthocyanins occur in red onions.29 To date, several structural and regulatory

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genes involved in the onion flavonoid pathway have been identified. Genes encoding

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chalcone synthase A and B (CHS-A and CHS-B), chalcone isomerase (CHI),

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dihydroflavonol 4-reductase (DFR), and ANS were identified through crossings between

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onion cultivars of different colors, with the resulting hybrids showing co-segregation of the

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respective

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(sequence-characterized amplified region) analyses with sets of Allium fistulosum (Japanese

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bunching onion)-shallot (Allium cepa L. Aggregatum group showing reddish-yellow sheaths)

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monosomic addition lines identified the chromosomal locations of the flavonoid biosynthesis

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genes, including CHS-A and CHS-B, CHI, F3H, flavonoid 3'-hydroxylase (F3'H), FLS, DFR,

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ANS, and UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT).43,44 CHS-A and CHS-B

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share 87% similarity at the amino acid level and are located on different chromosomes. 38,43

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The SCAR analyses showed that CHS-A was present only in shallot chromosome, while

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CHS-B was present in A. fistulosum as well as shallot chromosome.43 The flavonoid

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measurements of A. fistulosum-shallot multiple alien addition lines demonstrated the

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functionality of F3'H and UFGT in flavonoid biosynthesis but the correlation between

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flavonol contents and the presence of FLS allele was elusive.44 Further investigations into

alleles

with

bulb

color

phenotypes. 38-42


PCR-based

SCAR


110

the enzymatic properties of the structural genes, their regulatory mechanisms, and the

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contents of related metabolites are still required for a complete understanding of flavonoid

112

biosynthesis in onions.

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In the present study, we isolated cDNAs of onion FLSs (AcFLSs) from a doubled-haploid

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line of red onion, ‘H6’, and the commercial yellow onion cultivar ‘Hwangryongball’

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(‘HRB’). Recombinant proteins from both AcFLS cDNAs were expressed in E. coli as

116

fusions with glutathione S-transferase (GST), and their enzymatic properties indicated that

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AcFLS-H6 and -HRB are functional enzymes that likely play a key role in flavonol

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biosynthesis. Moreover, our expression analyses of flavonoid biosynthetic genes and the

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quantitative measurements of flavonols and anthocyanins in different tissues provide

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valuable information about the flavonoid biosynthetic pathway in onion.

121 122 123

Materials and Methods

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Plant materials: This study used the red onion ‘H6’ (a doubled-haploid line) and the

125

yellow onion ‘Hwangryongball’ (HRB; developed from a Korean landrace). The H6 line

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was induced by whole-flower culture through in vitro gynogenesis to avoid the

127

heterozygosity resulting from cross-pollination; thus, the doubled-haploid in H6 is

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completely homozygous for all loci.40 HRB is a mid-late maturing commercial cultivar, and

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the outer scale color of its mature bulb is deep yellowish brown. Tissue samples, e.g.,

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leaves, sheaths, inner bulbs, and roots, were prepared from 3-month-old seedlings of H6

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and HRB grown in a greenhouse. The sheath and bulb parts of the seedlings were red in H6, ACS Paragon Plus Environment

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but white in HRB. However, the red color appeared on only one or two layers of the sheath

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and bulb of H6. Therefore, we peeled two layers from both onions and designated them as the

134

‘sheath’, and the remaining part of the bulb was designated as the ‘inner bulb’. After sample

135

collection, the tissues were immediately frozen in liquid nitrogen and stored at -80 °C until

136

total RNA and flavonol extraction.

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Chemical standards: (±)-Dihydrokaempferol, (±)-dihydroquercetin, keampferol, and

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quercetin were purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO), and

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Cyanidin chloride was purchased from Extrasynthese (Extrasynthese, Genay Cedex,

140

France). The dihydroflavonols and flavonols were prepared as stock solution at 100 mM in

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dimethyl sulfoxide, whereas cyanidin chloride was prepared at 100 mM in 50% methanol

142

containing 1.2 N HCl. UV external standard calibration was carried out to obtain

143

calibration curves of kaempferol, quercetin, and cyanidin-chloride, which were used to

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quantify flavonol aglycones and glucosides, and cyanidin aglycones and glucosides.

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Total RNA extraction and cloning of AcFLS genes: The frozen tissue samples (100

146

mg) were ground to a powder in liquid nitrogen using a mortar and pestle and total RNA

147

was extracted with Fruit-mate for RNA Purification solution (Takara, Otsu, Japan) and Plant

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RNA Purification Reagent (Invitrogen, Carlsbad, CA) as described previously. 45 cDNAs

149

were synthesized with 2 μg of total RNA and amfiRivert cDNA Synthesis Platinum Master

150

Mix (GenDEPOT, Barker, TX) according to the manufacturer’s instructions. The first-strand

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cDNAs synthesized from the total RNA of the sheaths of H6 and HRB were used as

152

templates for PCR amplification of the AcFLS cDNAs. The gene-specific primers were

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designed based on the cDNA sequence of the FLS allele of H6 previously deposited into ACS Paragon Plus Environment


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GenBank (AY221247).40 The primer sequences used for the amplification of the open

155

reading

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5'-ATGGAAGTAGAGAGAGTGCAGGC-3';

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5'-TTACTGAGGAAGTTTATTAATTTTGC-3'. PCR was performed using PrimeSTAR

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HS DNA polymerase (Takara) under the following conditions: 98 °C for 2 min; 32 cycles of

159

98 °C for 10 s, 55 °C for 15 s, and 72 °C for 1 min 10 s; and a final extension at 72 °C for 3

160

min. The PCR-amplified fragments of the AcFLS isolated from H6 (AcFLS-H6) and from

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HRB (AcFLS-HRB) were ligated into the pTOP Blunt V2 vector (Enzynomics, Daejeon,

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Korea) to generate pTOP Blunt V2:AcFLS-H6 and pTOP Blunt V2:AcFLS-HRB, and

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verified by sequencing. To clone the AcFLSs into the bacterial expression vector

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pGEX-4T-3, the coding regions of the AcFLSs were amplified by PCR using PrimeSTAR

165

HS DNA polymerase (Takara). The pTOP Blunt V2:AcFLS-H6 and pTOP Blunt

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V2:AcFLS-HRB were used as templates and primers were designed to contain the regions

167

complementary to the termini of the insert and the pGEX-4T-3 vector linearized by BamHI

168

(Forward,

169

5'-GGGAATTCGGGGATCCTTACTGAGGAAGTTTA- 3'). PCR was performed under

170

the following conditions: 98 °C for 30 s; 32 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C

171

for 1 min 10 s; and a final extension at 72 °C for 3 min. The PCR products were cloned into

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the pGEX-4T-3 vector linearized by BamHI digestion using In-Fusion Advantage PCR

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Cloning Kits (Clontech, Mountain View, CA) in frame with the sequence encoding the

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N-terminal glutathione-S-transferase (GST) tag. The resulting pGEX-4T-3:AcFLS-H6 and

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pGEX-4T-3:AcFLS-HRB vectors were subsequently sequenced.

frames

(ORF)

of

the

AcFLSs

were

as

follows:

5'-GGTTCCGCGTGGATCCATGGAAGTAGAGAGAG-3';


Forward, Reverse,

Reverse,

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Expression and purification of AcFLS-HRB and AcFLS-H6 in E. coli: The

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pGEX-4T-3:AcFLS-H6 and pGEX-4T-3:AcFLS-HRB vectors were transformed into E. coli

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strain BL12 (DE3) (Novagen, Darmstadt, Germany). Protein expression was induced at

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24 °C for 6 h in the presence of isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.2 mM) in

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50 mL LB medium. Bacterial cells were then harvested and sonicated in 2 mL lysis buffer (50

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mM sodium phosphate, pH 8.0), 150 mM NaCl, 1% triton X-100 (v/v), 10% glycerol (v/v), 2

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mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). After centrifugation (13,000 g,

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4°C, 10 min), 100 μL Glutathione Sepharose 4B beads (GE Healthcare, Pittsburgh, PA)

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were added to the soluble bacterial lysate and incubated at 4 °C for 2 h with gentle rotation.

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The beads were collected, washed four times with 1 × PBS (137 mM NaCl, 2.7 mM KCl,

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100 mM Na2HPO4, and 2 mM K2HPO4, pH 7.4), and then eluted three times with one bead

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volume of elution buffer (50 mM Tris-Cl, pH 8.0) and 10 mM reduced glutathione). The

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purified recombinant proteins were quantified by the Bradford method46 and verified by

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SDS-PAGE.

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In vitro assay for FLS activity: The enzymatic activities of GST-AcFLS-H6 and

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GST-AcFLS-HRB were assayed in a 100-μL reaction containing 50 mM glycine-NaOH

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(pH 8.5), 10 mM 2-oxoglutarate (disodium salt), 0.25 mM ferrous sulfate, and 2-400 μM

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dihydrokaempferol or dihydroquercetin as a substrate. Reactions were initiated by the

194

addition of substrate and incubated at 25 °C for 10 min; then, 100 μL ethyl acetate was

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added to the reactions and mixed vigorously for 1 min. After centrifugation, 50 μL ethyl

196

acetate layers were evaporated and the residues were dissolved in 100 μL methanol for

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high-performance liquid chromatography (HPLC) injection. HPLC was performed on an ACS Paragon Plus Environment


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LC-20A HPLC system (Shimadzu, Kyoto, Japan) connected with an Inertsil-ODS3 C18

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column (5 μm, 250 × 4.6 mm, GL Science). The mobile phase consisted of 0.1% formic acid

200

(A) and acetonitrile containing 0.1% formic acid (B). The gradient profile was optimized as

201

follows: 0 min, 95% A/5% B; 30 min, 45% A/55% B; 45 min, 35% A/65% B; 50 min, 0%

202

A/100% B; 52 min, 95% A/5% B; 60 min, 95% A/5% B. The flow rate was 1 mL∙min-1 and

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the temperature of the column was maintained at 40 °C. A diode-array detector was used for

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real-time monitoring of the chromatograms. The flavonol products kaempferol and quercetin

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were identified by comparing the retention times and UV-VIS spectra recorded from 210 nm

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to 800 nm with those of standards.

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Gene expression analysis: cDNAs prepared from the leaves, sheaths, inner bulbs, and

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roots were used as templates for the amplifications of structural genes of the flavonoid

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biosynthetic pathway using quantitative PCR (qPCR). qPCR was performed in 15-μL

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reactions containing AccuPower 2 × Greenstar qPCR Master Mix (Bioneer, Daejun, Korea)

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and 0.4 μM each primer (Table 1). Data were normalized to the expression levels of

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ß–Tubulin (AcTUB) (accession number: AA451549). qPCR was carried out on a BioRad

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CFX96 Detection System (Bio-Rad Laboratories, Hercules, CA) under the following

214

conditions: 95 °C for 15 min followed by 40 cycles of 95 °C for 15 s and 55 °C for 30 s. The

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amplification specificities were verified by melting curve analyses (55 °C-95 °C) and each

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sample was run in triplicate.

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Determination of flavonoid glucosides and aglycones: The ground onion tissues were

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lyophilized and 0.05 g of each sample was mixed with 600 μL MFW solution

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(methanol:formic acid:water; 50:5:45; v/v/v) optimized for flavonoid extraction. 32,33 The ACS Paragon Plus Environment

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mixed solutions were incubated at room temperature for 1 h with shaking and subsequently

221

centrifuged at 15,000 g at 4 °C for 10 min. Additional extractions with 10 min incubations

222

were repeated twice and each extract solution was combined. The extracts were filtered

223

through 0.45 μm Teflon polytetrafluoroethylene (PTFE) syringe filters, and then diluted

224

10-fold with MFW solution prior to ultra-performance liquid chromatography (UPLC)

225

injection. Acid hydrolysis was carried out to convert flavonoid glucosides into the

226

respective aglycones. The aliquots of the filtered extracts were diluted 10-fold with MFW

227

solution containing 3 N HCl, and then incubated at 90 °C for 2 h. After centrifugation

228

(15,000 g, 4 °C, 5 min), the supernatants were subjected to flavonoid analysis. The

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individual flavonoid aglycones and glycosides were analyzed using an ultra-performance

230

liquid chromatography-diode array detector-electrospray ionization-quadrupole time of

231

flight mass spectrometry (UPLC-DAD-ESI-QTOF/MS) system (Waters MS Technologies,

232

Manchester, UK). The separation was performed with an Luna Omega 1.6 μm column (C18,

233

150 × 2.1 mm) (Phenomenex, Torrance, CA) and SecurityGuard TM ULTRA cartridges C18

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for 2.1 ID pre-column (Phenomenex) operated at a temperature of 35 ℃. The mobile phase

235

consisted of 0.5% formic acid in water (A) and 0.5% formic acid in acetonitrile (B) at a

236

flow of 0.3 mL∙min-1 using the following gradient program: 0 min, 7% B; 2 min, 7% B; 24

237

min, 15% B; 40 min, 30% B; 48 min, 60% B; 50 min, 60% B; 53 min, 90% B; 54 min, 90%

238

B; 55 min, 7% B; 60 min, 7% B. The injection volume for all samples was 5 μL. Specific

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wavelengths were monitored separately at 288 nm for flavanones and dihydroflavonols, and

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350 nm for flavones and flavonols, and 520 nm for anthocyanins. The UPLC was coupled

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to a Xevo G2-S QTOF-ESI/MS (Waters). The scan range was m/z 50 – 800 in positive ion ACS Paragon Plus Environment


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mode. The capillary and sampling cone voltages were 3.5 kV and 40 V, respectively. The

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desolvation gas was maintained at 1050 L∙h-1 at a temperature of 500 ℃. The cone gas was

244

maintained at 50L∙h-1 with a ion source temperature of 120 ℃. Data acquisition and

245

processing were performed using Waters MassLynx version 4.1 software.

246 247 248

Results

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The cDNAs of AcFLS-H6 and AcFLS-HRB encode FLS enzymes: The sequence of a

250

putative FLS cDNA from the red onion doubled-haploid line H6 was previously deposited in

251

GenBank under accession no. AY221247.40 Another putative FLS sequence (AY647262)

252

was identified from shallot by SCAR analysis of A. fistulosum-shallot monosomic addition

253

lines and was assigned to chromosome 4A. 44 Sequence alignment showed that these two

254

sequences shared 99.2% identity at the nucleotide level. Thus, we regarded these sequences

255

as allelic variants. We isolated AcFLS sequences containing 1,008 bp ORFs from H6 and

256

HRB with primers designed from the AY221247 sequence. Unexpectedly, the isolated

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cDNA sequence from H6 was different from the AY221247 sequence at the 61, 284, 333,

258

and 418 bp positions (Figure S1), and the resulting deduced amino acid sequence was

259

different at the amino acid positions 21, 95, and 140. This result was confirmed by

260

sequencing the FLS cDNAs of H6 from three independent reverse transcription-PCR

261

reactions. Thus, we designated this sequence as AcFLS-H6 (deposited in GenBank with

262

accession KY369209). The FLS cDNA sequence from HRB was different from the

263

AcFLS-H6 sequence at four nucleotide positions (Figure S1), which resulted in different ACS Paragon Plus Environment

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amino acids at positions 45, 65, and 213 (Figure 2). This sequence was designated as

265

AcFLS-HRB (GenBank accession KY369210). The deduced AcFLS-H6 and AcFLS-HRB

266

proteins contained 335 amino acids and their molecular weights were approximately 38 kDa,

267

with isoelectric points of 6.3 for AcFLS-H6 and 6.1 for AcFLS-HRB.

268

When the two AcFLSs were aligned with other plant 2-ODD proteins with known

269

functions, both AcFLS proteins were found to possess conserved HxDxnH motifs (His221,

270

Asp223, and His277) for binding ferrous iron and RxS motifs (Arg287 and Ser289) for

271

binding 2-oxoglutarate. The FLS sequences, including the AcFLSs, were more similar to the

272

ANS sequences than to those of F3H or FNS, particularly in the third conserved region of

273

2-ODD proteins (Figure 2). The alignment also showed that the ‘PxxxIRxxxEQP’ and

274

‘SxxTxLVP’ motifs, which have been proposed to be FLS-specific regions distinct from

275

other plant 2-ODDs,47 were conserved in both AcFLS sequences and in the other plant FLSs

276

(Figure 2). The putative substrate-binding residues of the AcFLSs (Tyr132, Phe134, Lys202,

277

Phe293, and Ser295) were assigned based on structural modeling of Arabidopsis thaliana

278

FLS.48 Of these amino acid residues, Phe134, Lys202, and Phe293 were conserved among all

279

FLS members. However, the residues corresponding to the Tyr132 and Ser295 positions in

280

the AcFLS sequences varied among the FLS members. Residues corresponding to Gly68 and

281

Gly261 of the AcFLSs, which may confer flexibility for proper folding,18 were identical

282

across all 2-ODD proteins examined (Figure 2). As mentioned above, three residues differed

283

between AcFLS-H6 and AcFLS-HRB, of which position 65 was located near the first

284

conserved region of the 2-ODDs, and position 213 was located in the second conserved

285

region of the 2-ODDs containing the HxD residues of the HxDxnH motif.18,47 The 213th ACS Paragon Plus Environment


286

residue of AcFLS-H6 was Asn, with a polar uncharged side chain, but the corresponding

287

residues of AcFLS-HRB and most of the other FLSs were Asp or Glu, which have negatively

288

charged side chains (Figure 2).

289

A phylogenetic tree was constructed with the functionally characterized plant 2-ODDs as

290

well as various putative 2-ODD sequences (Figure 3). Both AcFLS sequences clustered with

291

other FLSs and were placed between the FLSs of monocots and eudicots. Two putative

292

2-ODD sequences (PP1S46_151V6.1 and PP1S287_51V6.2) from mosses (Physcomitrella

293

patens) identified by a homology search with AcFLSs were placed along with these 2-ODDs

294

in the phylogenetic tree, which suggests that the occurrence of FLS was a relatively early

295

event in the evolution of plants. Indeed, mosses have been found to contain the flavonoids

296

flavone C- and O-glycosides, biflavones, aurones, isoflavones, and 3-deoxyanthocyanins.49

297

Collectively, our sequence analyses clearly indicated that the AcFLS-H6 and AcFLS-HRB

298

genes encode an FLS enzyme.

299

Recombinant AcFLS-H6 and AcFLS-HRB proteins show FLS activity: We

300

constructed GST-fused recombinant proteins of AcFLS-H6 and AcFLS-HRB to determine

301

whether they harbored FLS activity. After induction of expression with IPTG, the

302

recombinant proteins were found among the total proteins of bacterial lysates. Most of the

303

GST-AcFLS protein was in the soluble fraction and the proteins were successfully purified

304

via GST affinity chromatography (Figure 4A). The FLS activity of the purified recombinant

305

proteins was assayed using dihydrokaempferol and dihydroquercetin as substrates in the

306

presence of ferrous iron and 2-oxoglutarate, and the products were analyzed by HPLC. As


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shown in Figure 4B, both recombinant proteins catalyzed the formation of kaempferol and

308

quercetin from dihydrokaempferol and dihydroquercetin, respectively.

309

We conducted kinetic analysis to determine the enzymatic properties of the two

310

recombinant AcFLS proteins, which revealed that both AcFLSs showed slightly lower Km

311

values for dihydrokaempferol (GST-AcFLS-H6: 20.3 ± 1.8 μM; GST-AcFLS-HRB: 15.5 ±

312

1.4 μM) than for dihydroquercetin (GST-AcFLS-H6: 24.4 ± 1.6 μM; GST-AcFLS-HRB:

313

26.3 ± 0.2 μM). For both AcFLSs, significantly higher turnover numbers (Kcat(s-1)) were

314

measured for dihydroquercetin (GST-AcFLS-H6: 0.0096 ± 0.00056; GST-AcFLS-HRB:

315

0.0211 ± 0.00082) than for dihydrokaempferol (GST-AcFLS-H6: 0.0014 ± 0.00004;

316

GST-AcFLS-HRB: 0.0024 ± 0.0001). Consequently, the catalytic efficiencies (Kcat/Km) of

317

GST-AcFLS-H6 and GST-AcFLS-HRB for dihydroquercetin were 5.7- and 5.2-fold higher,

318

respectively, than those for dihydrokaempferol. When comparing the two AcFLSs, the

319

turnover numbers of GST-AcFLS-HRB for dihydrokaempferol and dihydroquercetin were

320

1.7- and 2.2-fold higher, respectively, than those of GST-AcFLS-H6 for the same substrates

321

(Table 2). These results indicated that both AcFLSs are functional proteins with FLS

322

activity and that they prefer dihydroquercetin to dihydrokaempferol as a substrate. In

323

addition, the FLS activity of GST-AcFLS-HRB was higher than that of GST-AcFLS-H6

324

with both of the dihydroflavonol substrates.

325

The expression levels of flavonoid biosynthetic genes vary in different tissues and

326

cultivars: The expression levels of five early biosynthesis genes (EBGs), CHS-A, CHI,

327

F3H, F3'H, and FLS, and two late biosynthesis genes (LBGs), DFR and ANS were analyzed

328

in different tissues of H6 and HRB. The sheath of the red onion, H6, showed red coloration ACS Paragon Plus Environment


329

attributed to anthocyanin accumulation,50 while the sheath of the yellow onion, HRB, was

330

white (Figure 5B). Gene expression analysis showed that most of the genes mentioned above

331

were predominantly expressed in the sheaths of both onions. When comparing gene

332

expression levels in the sheaths of H6 and HRB, the levels of CHS-A and F3'H in H6 were

333

approximately 2.5- and 1.5-fold higher, respectively, than those in HRB, and the levels of

334

F3H and ANS in H6 were approximately 14-fold higher than those in HRB. DFR expression

335

was detected only in H6, whereas FLS were expressed at almost same levels in both onions

336

(Figure 5A). The absence of the DFR transcript in HRB is consistent with the previous

337

finding that yellow onions carry a mutant DFR allele that cannot be transcribed into an intact

338

mRNA. This finding corresponds to the different coloration in the sheaths between H6 and

339

HRB as shown in Figure 5B. In the other tissues, the levels of CHS-A, F3H, and ANS were

340

consistently higher in H6 than in HRB, and the levels of CHI and FLS were higher in the

341

leaves of H6 than in the leaves of HRB, but were similar in the inner bulbs and roots of both

342

onions. Contrary to the higher gene expression levels found in most tissues of H6, the level of

343

F3'H was higher in the inner bulb of HRB than that of H6, but its expression in the leaves and

344

roots was higher in H6. Overall, the expression levels of flavonoid biosynthetic genes were

345

higher in H6 than in HRB, suggesting that flavonoid biosynthesis and B-ring hydroxylation

346

of flavonoids are more active in H6 compared to HRB at this growth stage, and indicating

347

that the genes are differentially regulated depending on the cultivar and tissue.

348

Flavonols and anthocyanins are highly accumulated in the sheaths of onions: To

349

measure the total amount of the aglycones of the each class of flavonols and anthocyanins in

350

the different tissues of the 3-month-old seedlings of H6 and HRB, the acid hydrolyzed ACS Paragon Plus Environment

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351

extracts were separated by UPLC (Figure 6A). Flavonols exhibit high absorbance at

352

240280 nm and at 300380 nm, while anthocyanins show absorbance maxima at 240280

353

nm and at 465560 nm.51 Peaks corresponding to the flavonols and anthocyanins were

354

identified according to the spectral characteristics and the QTOF/MS analysis (Figure S2).

355

The areas of peaks were measured at 350 nm and 520 nm for flavonols and anthocyanins,

356

respectively (Figure 6). Large amounts of quercetin aglycones were detected in the sheaths

357

of both onions. The quercetin level in the sheath of H6 (65.3 ± 5.5 μmol g-1 DW) was

358

approximately 1.5-fold higher than that of HRB (44.6 ± 2.7 μmol g-1 DW), which is in

359

agreement with the previous literature showing that red onions contain higher amounts of

360

flavonols than yellow onions.27 Small quantities of quercetin were observed in the leaves and

361

inner bulbs. The levels of quercetin in the leaves were similar in both onions (0.85 ± 0.06

362

μmol g-1 DW in H6; 0.95 ± 0.03 μmol g-1 DW in HRB), whereas 7.8-fold more quercetin was

363

found in the inner bulb of HRB compared to that of H6 (0.14 ± 0.01 μmol g-1 DW in H6; 0.08

364

± 0.004 μmol g-1 DW in HRB). Trace levels of quercetin were also observed in the roots of

365

both onions (0.31 ± 0.02 μmol g-1 DW in H6; 2.43 ± 0.1 μmol g-1 DW in HRB). Kaempferol

366

was detected only in the HRB leaf at trace levels (0.19 ± 0.015 μmol g-1 DW), whereas the

367

other samples did not contain detectable kaempferol. Isorhamnetin, a 3’-O-methylated

368

quercetin, was also detected in the sheaths of both onions, and the level in H6 was higher

369

than in HRB (2.73 ± 0.2 μmol g-1 DW in H6; 2.14 ± 0.15 μmol g-1 DW in HRB). The overall

370

amounts of the isorhamnetins were as low as approximately 4.5% of the overall quercetin

371

contents in the sheaths of both onions. Myricetin, a 3',4',5'-hydroxylated flavonol, was not

372

detected in any tissues of either onion, suggesting that flavonoid 3’,5’-hydroxylase activity ACS Paragon Plus Environment


373

is likely to be absent in these onions. Anthocyanin aglycone, shown as a single peak at 520

374

nm, was observed only in the sheath of H6 (Figure 6B). The QTOF/MS analysis of the peak

375

exhibited an [M]+ ion at m/z 287, corresponding to cyanidin (Figure S2). The cyanidin

376

content was 5.55 ± 0.6 μmol g-1 DW, reaching 8.5% of the quercetin content in the sheath of

377

H6, which indicates that cyanidins are the second-most abundant flavonoid in H6 (Figure

378

6C). Besides flavonols and anthocyanins, no peaks corresponding to other classes of

379

flavonoids, such as flavanones, flavones, dihydroflavonols, were detected in this study.

380

Individual flavonols and cyanidin glucosides vary between the two onions: The

381

accumulation patterns of flavonoid glucosides in the sheaths of both onions were analyzed

382

to understand the downstream of flavonoid metabolism. Sample extracts not subjected to

383

acid hydrolysis were separated by UPLC. Peaks at 350 nm and 520 nm were identified

384

based on the QTOF/MS analysis and published data. As shown in Figure 7A, H6 and HRB

385

sheaths contained two major (peak 1 and peak 4) and four minor (peak 2, peak 3, peak 5,

386

and peak 6) flavonols, and the H6 sheath also contained two major (peak 7 and peak 8) and

387

one minor (peak 9) anthocyanins. Peaks 1, 3, 4, and 6 had [M+H]+ ion at m/z 303,

388

corresponding to quercetin. Peak 1 showed an additional [M+H]+ ion at m/z 465, 627 due to

389

conjugation of two glucose moieties (162 atomic mass units (amu) + 162 amu), whereas

390

peaks 3 and 4 showed an additional [M+H]+ ion at m/z 465, indicating the conjugation of

391

one glucose moiety. Peaks 2 and 5 exhibited [M+H]+ ion at m/z 317, corresponding to

392

isorhamnetin. Peak 2 showed additional [M+H]+ ion at m/z 479, 641, and peak 5 showed an

393

additional [M+H]+ ion at m/z 479, indicating the conjugation of two and one glucose moiety,

394

respectively (Figure S3). According to the order of retention times of the flavonols reported ACS Paragon Plus Environment

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395

previously,32 peaks 1-6 were tentatively identified as quercetin 3,4'-diglucoside,

396

isorhamnetin 3,4'-diglucoside, quercetin 3-glucoside, quercetin 4'-glucoside, isorhamnetin

397

4'-glucoside, and quercetin, respectively. Peaks 7, 8, and 9 presented [M]+ at m/z 287,

398

which corresponds to cyanidin. Peak 7 also showed one glucose conjugation ([M]+ at m/z

399

449). However, peak 8 showed an additional [M]+ at m/z 535 consisting of 449 amu and 86

400

amu, i.e., a malonyl moiety, and peak 9 showed additional [M]+ at m/z 697 corresponding

401

to the conjugation of two glucoses and one malonyl moiety (Figure S4). Peaks 7-9 were

402

further tentatively identified as cyanidin 3-glucoside, cyanidin 3-(6''-malonylglucoside),

403

and cyanidin 3-(6''-malony-laminaribioside), respectively, according to the previous

404

report.32 The quantities of the individual flavonols and cyanidins in the sheaths of both

405

onions were compared (Figure 7B). Most quercetins were glycosylated, leaving trace

406

residues of quercetin aglycones in both samples. Quercetin 4'-glucoside was the most

407

abundant flavonol, followed by quercetin 3,4'-diglucoside. The content of quercetin

408

4'-glucoside in H6 was 1.7-fold higher than that in HRB. However, the amounts of

409

quercetin 3,4'-diglucoside were similar between H6 and HRB. The main anthocyanin in the

410

extracts was cyanidin 3-(6''-malonylglucoside), which accounted for 85% of total amount of

411

anthocyanins in the sheath of H6.

412 413 414

Discussion

415

Several genes involved in flavonoid biosynthesis in onions have been identified so far.

416

However, FLS, an important gene for flavonol biosynthesis, has not been functionally ACS Paragon Plus Environment


417

characterized in onion. In contrast to the identification of the other structural genes through

418

crossing based on the inheritance of bulb color, identification of FLS has been hindered by

419

the fact that flavonols have only a minor effect on coloration. Functional redundancy

420

caused by other genes that exhibit similar functions and the involvement of flavonols in

421

male sterility may also have been barriers to the identification of FLS through crossing.

422

Consequently, only the chromosomal location of one putative AcFLS sequence has been

423

assigned thus far. It is not yet clear whether there are other AcFLS isogenes positioned at

424

different loci.

425

Here, we described the isolation and characterization of AcFLS sequences from H6 and

426

HRB onions and demonstrated that these two sequences are genes encoding FLS enzymes

427

capable of converting dihydroquercetin and dihydrokaempferol to quercetin and kaempferol,

428

respectively.

429

Within the 2-ODD protein family, FLS and ANS are grouped together with a high degree

430

of sequence similarity. Some FLS and ANS enzymes show multiple functions; for instance,

431

the additional F3H activity of FLS and the additional FLS activity of ANS. This

432

multifunctionality indicates that these enzymes have broad substrate specificities, whereas

433

FNSI and F3H, forming a separate group, show narrow substrate specificities. 52-54 Among

434

the five amino acid residues of the AcFLS proteins that are predicted to be responsible for

435

substrate binding, Tyr132 and Ser295 are not conserved across all FLS members. At the

436

position corresponding to the Tyr132 residue of the AcFLS proteins, CsFLS has the same Tyr

437

residue, but CuFLS, FtFLS, and AtFLS have a His residue and ZmFLS and GbFLS have a

438

Phe residue. When the His residue of AtFLS was replaced with Tyr or Phe by site-directed ACS Paragon Plus Environment

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439

mutagenesis, the Km values for dihydroquercetin decreased by approximately 50%.48 Thus,

440

the residues containing a phenyl ring at this position, such as the Tyr132 of AcFLSs or the

441

Phe residue of ZmFLS and GbFLS, might have a positive effect in terms of dihydroquercetin

442

binding compared to other FLSs possessing a His residue at this position.

443

There are three amino acid variations between AcFLS-H6 and AcFLS-HRB. Two of the

444

three variations are located at the N-terminal variable region, whereas the other variation at

445

position 213 is in the center of the second highly conserved region containing the HxD

446

ferrous iron-binding residues. Position 213 of AcFLS-H6 is an Asn residue having a polar

447

uncharged side chain, while the residues in the corresponding position of most of the other

448

FLSs, including AcFLS-HRB, are acidic residues such as Asp and Glu. Such an amino acid

449

change at position 213 may affect the protein secondary structure, which may cause subtle

450

conformational changes in the adjacent active site in which interaction between HxDxnH

451

motif and iron takes place. Our kinetic analysis of the recombinant AcFLS-H6 and

452

AcFLS-HRB revealed that there were no significant differences in Km values between the

453

two recombinant proteins for each substrate; however, the turnover numbers of AcFLS-HRB

454

were approximately 2-fold higher than those of AcFLS-H6 for both dihydrokaempferol and

455

dihydroquercetin (Table 2). This difference between the two AcFLSs seems likely to be

456

caused by the difference at the three amino acids, especially by the position 213 variation.

457

Both recombinant

458

dihydroquercetin than for dihydrokaempferol. By contrast, previous reports demonstrated

459

that AtFLS1, ZmFLS, and CuFLS showed higher preferences for dihydrokaempferol than

460

for dihydroquercetin.18,22,55 Given that quercetin is the most abundant flavonoid in onions,

AcFLSs

showed

significantly higher


turnover

numbers

for


461

this substrate preference of the AcFLSs could be an advantage for the production of

462

quercetin.

463

We analyzed the expression of flavonoid biosynthesis genes and flavonol contents in

464

different tissues of H6 and HRB to better understand flavonoid biosynthesis in onion. The

465

expression of CHSs, which encodes the enzyme for the first committed step in flavonoid

466

biosynthesis, has been reported to be controlled by a regulator encoded by the C locus. The C

467

locus in white onion is suggested to be mutated so that significantly reduced expression of

468

CHS-A and CHS-B is observed in white onion.38 Our study showed that most flavonoid

469

biosynthetic pathway genes, including CHS-A, are expressed at remarkably lower levels in

470

the sheath of HRB compared to H6. Although the C locus in yellow onion has yet to be

471

identified, it is clear that there are differences in regulation patterns of flavonoid pathway

472

between H6 and HRB. The expression levels of the EBGs, such as CHS-A and F3H, are

473

considerably lower in the sheaths of HRB compared to H6. Even though CHS-B expression

474

was not examined here, it is plausible that the metabolic flow in the upstream portion of the

475

pathway is highly enhanced in H6 sheath compared to HRB sheath. Nevertheless, the

476

difference in quercetin levels between H6 and HRB sheath was smaller than our

477

expectation. It is possible that AcFLS-HRB exhibits higher FLS activity compared to

478

AcFLS-H6, contributing to the flavonol production in the sheath of HRB. In addition,

479

dihydroquercetin, a substrate for FLS, in H6 sheath might be consumed as a substrate by

480

DFR present only in H6, which might also contribute to the reduction of quercetin in the

481

sheath of H6. Furthermore, F3’H could be a critical determinant for the accumulation

482

pattern of flavonol. Previous reports suggested the possibility that flavonoid biosynthesis ACS Paragon Plus Environment

Page 22 of 46

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483

takes place within a metabolon complex composed of CHS, F3H, F3’H, DFR and ANS, in

484

which F3’H, a membrane-bound cytochrome P450, may anchor the complex to the surface

485

of the endoplasmic reticulum.56,57 If so, the level of F3’H protein could determine the

486

amount of the metabolon complex present. Indeed, the expression pattern of F3’H in H6

487

and HRB (Figure 5A) is similar to the accumulation pattern of quercetin in both onions

488

(Figure 6C). The level of quercetin in HRB inner bulb is significantly higher than that in

489

H6 inner bulb, which seems to be caused by the higher level of F3’H expression in the

490

HRB inner bulb; moreover, the enhanced activity of AcFLS-HRB could contribute to

491

widen the gap between the quercetin levels in H6 and HRB inner bulbs.

492

The absence of kaempferol in the sheaths of both onions indicates that F3'H activity was

493

sufficient to completely convert 4'-hydroxylated dihydrokaempferol or kaempferol into

494

3',4'-hydroxylated dihydroquercetin or quercetin, respectively. We found small amounts of

495

isorhamnetins in sheaths, and the accumulation pattern was in accordance with the pattern

496

of quercetin in sheaths, suggesting that quercetin 3'-O-methyltransferase activity is similar

497

between H6 and HRB sheaths. The level of cyanidin accounted for only 8.5% of the

498

quercetin in H6 sheath. This result implies that FLS dominates the competition between

499

FLS and DFR for their substrate dihydroquercetin. Unlike most of the other genes examined,

500

the expression of FLS in HRB sheath is maintained at a similar level to that of H6. Although

501

HRB seems to be unfavorable to producing large amount of flavonoids compared to H6, the

502

maintained expression of FLS and the enhanced FLS activity could maximize flavonol

503

production in HRB sheath, which may compensate for deficiencies of some physiological

504

roles that might be caused by the absence of anthocyanins in the HRB. ACS Paragon Plus Environment


505

In general, the most prevalent flavonoid in onion bulbs is quercetin 4'-glucoside, which is

506

believed to be formed first, and followed by quercetin 3,4'-diglucoside formation from the

507

quercetin 4'-glucoside during maturation and further storage.32,58 Correspondingly, the level

508

of quercetin 4'-glucoside is the highest in the sheaths of both onions (Figure 7B).

509

Furthermore, the level of quercetin 4'-glucoside in H6 is much higher than that in HRB,

510

which is in agreement with the accumulation pattern of quercetin aglycone, implying that

511

quercetin 4'-O-glucosyltransferase activities would be similar in both onions. By contrast,

512

quercetin 3,4'-diglucoside levels in both onions are similar, indicating that quercetin

513

3-O-glucosyltransferase activity in HRB is likely higher than that in H6.

514

Taken together, the AcFLS proteins we characterized are authentic flavonol synthases in

515

onions, and our analyses of gene expression and flavonol contents provide insight into the

516

flavonoid biosynthesis in onion.

517 518 519

Acknowledgments

520

We are grateful to Prof. Sunggil Kim (Chonnam National University) for providing the seeds

521

of H6 onion. We are also grateful to NHSEED (Nonghyup Inc.), for providing the seeds of

522

HRB onion.

523 524 525

Supporting Information Available:


Page 24 of 46

Page 25 of 46


526

Figure S1. Sequence comparison of AY221247, AcFLS-H6, and AcFLS-HRB cDNAs. The

527

identical nucleotides at positions showing variations are indicated by black rectangles.

528

Figure S2. The mass and UV spectra of quercetin (Q) in HRB sheath, kaempferol (K) in

529

HRB leaf, isorhamnetin (IR) in H6 sheath, and cycanidin (C) in H6 sheath.

530

Figure S3. The mass spectra of the compounds corresponding to peak 1-6.

531

Figure S4. The mass spectra of the compounds corresponding to peak 7-10.

532 533 534

Funding sources

535

This work was supported by a fund from the National Institute of Agricultural Science

536

(PJ012458201701) and a grant from the Next-Generation BioGreen 21 Program

537

(PJ011094201702), Rural Development Administration, Republic of Korea.

538 539 540

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30. Rodrigues, A. S.; Pérez-Gregorio, M. R.; García-Falcón, M. S.; Simal-Gándara, J.

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Effect of curing and cooking on flavonols and anthocyanins in traditional varieties of

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31. Rodrigues, A. S.; Pérez-Gregorio, M. R.; García-Falcón, M. S.; Simal-Gándara, J.;

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onion cultivars. Food Control 2010, 21, 878–884.

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32. Pérez-Gregorio, M. R.; García-Falcón, M. S.; Simal-Gándara, J.; Rodrigues, A. S.;

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Almeida, D.P. F. Identification and quantification of flavonoids in traditional cultivars

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of red and white onions at harvest. J. Food Comp. Anal. 2010, 23, 592-598.

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33. Pérez-Gregorio, González-Barreiro, C.; Rial-Otero, R.; Simal-Gándara, J. Comparison

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34. Pérez-Gregorio, M. R., García-Falcón, M. S. and Simal-Gándara, J. (2011a).

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Flavonoids changes in fresh-cut onions during storage in different packaging systems.

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Food Chem. 2011, 124, 652–658.

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35. Suzuki, H.; Sawada, S.; Watanabe, K.; Nagae, S.; Yamaguchi, M.; Nakayama, T.;

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Nishino,

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malonyltransferase from scarlet sage (Salvia splendens) flowers: an enzyme that is

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phylogenetically separated from other anthocyanin acyltransferases. Plant J. 2004, 38,

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994–1003.

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36. Pérez-Gregorio, M. R.; Regueiro, J.; González-Barreiro, C.; Rial-Otero, R.;

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Simal-Gándara, J. Changes in antioxidant flavonoids during freeze-drying of red

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onions and subsequent storage. Food control 2011, 22, 1108-1113.

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37. Holton, T. A.; Cornish, E. C. (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 1995, 7, 1070-1083.



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38. Kim, S.; Yoo, K. S.; Pike, L. M. The basic color factor, the C locus, encodes a

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regulatory gene controlling transcription of chalcone synthase genes in onions (Allium

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cepa). Euphytica 2005, 142, 273–282.

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39. Kim, S.; Jones, R.; Yoo, K. S.; Pike, L. M. Gold color in onions (Allium cepa): a natural

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mutation of the chalcone isomerase gene resulting in a premature stop codon. Mol.Genet.

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Genomics 2004, 272, 411-419.

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40. Kim, S.; Binzel, M. L.; Park, S.; Yoo, K. S.; Pike, L. M. Inactivation of DFR

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(Dihydroflavonol 4-reductase) gene transcription results in blockage of anthocyanin

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production in yellow onions (Allium cepa). Mol. Breed. 2004, 14, 253-263.

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41. Kim, S.; Binzel, M. L.; Yoo, K. S.; Park, S.; Pike, L. M. Pink (P), a new locus

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responsible for a pink trait in onions (Allium cepa) resulting from natural mutations of

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anthocyanidin synthase. Mol. Genet. Genomics 2004, 272, 18-27.

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42. Kim, S.; Jones, R.; Yoo, K. S.; Pike, L. M. The L locus, one of complementary genes

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required for anthocyanin production in onions (Allium cepa), encodes anthocyanidin

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synthase. Theor. Appl. Genet. 2005, 111, 120-127.

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43. Masuzaki, S.; Shigyo, M.; Yamauchi, N. Complete assignment of structural genes

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involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of

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the shallot (Allium cepa L.). Genes Genet. Syst. 2006, 81, 255-263.

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44. Masuzaki, S.; Shigyo, M.; Yamauchi, N. Direct comparison between genomic

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constitution and flavonoid contents in Allium multiple alien addition lines reveals

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chromosomal locations of genes related to biosynthesis from dihydrokaempferol to

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quercetin glucosides in scaly leaf of shallot (Allium cepa L.). Theor. Appl. Genet. 2006,

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112, 607-617.

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45. Park, S.; Choi, M. J.; Lee, J. Y.; Kim, J. K.; Ha, S. H.; Lim, S. H. Molecular and

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biochemical analysis of two rice flavonoid 3'-hydroxylase to evaluate their roles in

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flavonoid biosynthesis in rice grain. Int. J. Mol. Sci. 2016, 17, 1549. ACS Paragon Plus Environment

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46. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram

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quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976,

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47. Stracke, R.; De Vos, R. C.; Bartelniewoehner, L.; Ishihara, H.; Sagasser, M.; Martens, S.;

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Weisshaar, B. Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis

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thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta

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2009, 229, 427-445.

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48. Chua, C. S.; Biermann, D.; Goo, K. S.; Sim, T. S. Elucidation of active site residues of

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Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering

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flavonols. Phytochemistry 2008, 69, 66-75.

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49. Jiang, C.; Schommer, C. K.; Kim, S. Y.; Suh, D. Y. Cloning and characterization of

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chalcone synthase from the moss, Physcomitrella patens. Phytochemistry 2006, 67,

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2531-2540.

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50. Fossen, T.; Andersen, O. M.; Ovstedal, D, O.; Pedersen, A. T.; Raknes, A.

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Characteristic anthocyanin pattern from onions and other Allium spp. J. Food Sci. 1996,

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61, 703–706.

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51. Merken, H. M.; Beecher, G. R. Measurement of food flavonoids by high-performance liquid chromatography: a review. J. Agric. Food Chem. 2000, 48, 577-599.

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52. Martens, S.; Forkmann, G.; Britsch, L.; Wellmann, F.; Matern, U.; Lukacin, R.

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Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley.

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FEBS Lett. 2003, 544, 93-98.

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53. Gebhardt, Y. H.; Witte, S.; Forkmann, G.; Lukacin, R.; Matern, U.; Martens, S.

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Molecular evolution of flavonoid dioxygenases in the family Apiaceae. Phytochemistry

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2005, 66, 1273–1284.



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54. Gebhardt, Y. H.; Witte, S.; Steuber, H.; Matern, U.; Martens, S. Evolution of flavone

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synthase I from parsley flavanone 3s-hydroxylase by site-directed mutagenesis. Plant

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Physiol. 2007, 144, 1442–1454.

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55. Owens, D. K.; Alerding, A. B.; Crosby, K. C.; Bandara, A. B.; Westwood. J. H.; Winkel,

704

B. S. Functional analysis of a predicted flavonol synthase gene family in Arabidopsis.

705

Plant Physiol. 2008, 147, 1046-1061.

706

56. Shih, C. H.; Chu, H.; Tang, L. K.; Sakamoto, W.; Maekawa, M.; Chu, I. K.; Wang, M.;

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Lo, C. Functional characterization of key structural genes in rice flavonoid biosynthesis.

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Planta 2008, 228, 1043-1054.

709 710

57. Winkel, B. S. J. Metabolic channeling in plants. Annu. Rev. Plant Biol. 2004, 55, 85–107.

711

58. Hirota, S.; Shimoda, T.; Takahama, U. Tissue and spatial distribution of flavonol and

712

peroxidase in onion bulbs and stability of flavonol glucosides during boiling of the

713

scales. J. Agric. Food Chem. 1998, 46, 3497–3502.

714 715

Figure captions

716 717

Figure 1. Putative flavonoid biosynthesis pathway in Allium cepa. CHS-A and CHS-B,

718

chalcone synthase A and B; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR,

719

dihydroflavonol 4-reductase; F3’H, flavonoid 3'-hydroxylase; FLS, flavonol synthase; ANS,

720

anthocyanidin synthase; Q4’GT, quercetin 4'-O-glucosyltransferase; Q3GT, quercetin

721

3-O-glucosyltransferase; OMT, O-methyltransferase; GT, glucosyltransferase.

722


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723

Figure 2. Alignment of the deduced amino acid sequences of AcFLS-H6 and AcFLS-HRB

724

with other 2-ODDs of the flavonoid biosynthesis pathway. The alignment was generated

725

using the ClustalW program. Identical amino acids are indicated with a black background.

726

The amino acids that are more than 70% conserved are indicated with a dark grey

727

background, and those that are more than 55% conserved are indicated with a light grey

728

background. The three regions with high similarity between the 2-ODDs are underlined.

729

The two boxes represent FLS-specific motifs ‘PxxxIRxxxEQP’ and ‘SxxTxLVP’. Amino

730

acid residues responsible for binding ferrous iron (His221, Asp223, and His277) and

731

2-oxoglutarate (Arg287 and Ser289) are marked with black and grey arrows, respectively.

732

The predicted residues involved in DHQ binding are marked with black circles (Tyr132,

733

Phe134, Lys202, Phe293, and Ser295). The functional residues suggested to be involved in

734

proper folding of the FLS polypeptide are marked with asterisks (Gly68 and Gly261). The

735

three different residues between AcFLS-H6 and AcFLS-HRB are indicated by red

736

rectangles (position 45, 65, and 213).

737 738

Figure 3. Phylogenetic tree of 2-ODD enzymes involved in the flavonoid biosynthesis

739

pathway. The tree was generated based on the sequence alignment of selected 2-ODD

740

proteins and drawn by the MEGA6 program. In addition to AcFLS-H6 and AcFLS-HRB

741

(marked with black circles), the following sequences were analyzed: AtFLS1 and AtFLS3

742

(Arabidopsis thaliana), CsFLS (Camellia sinensis), CuFLS (Citrus unshiu), FtFLS

743

(Fagopyrum tataricum), GbFLS (Ginkgo biloba), GmFLS (Glycine max), NtFLS

744

(Nicotiana tabaccum), OsFLS (Oryza sativa), PhFLS (Petunia hybrida), SbFLS (Sorghum ACS Paragon Plus Environment


745

bicolor), StFLS (Solanum tuberosum), VvFLS (Vitis vinifera), ZmFLS (Zea mays), AcANS

746

(Allium cepa), AtANS (Arabidopsis thaliana), OsANS (Oryza sativa), PaANS (Phytolacca

747

americana), PhANS (Petunia hybrida), VvANS (Vitis vinifera), AgFNSI (Apium

748

graveolens), DcFNSI (Daucus darota), PcFNSI (Petroselinum crispum), AcF3H (Allium

749

cepa), AtF3H (Arabidopsis thaliana), GmF3H (Glycine max), OsF3H-1, OsF3H-2, and

750

OsF3H-3 (Oryza sativa), PP1S46_151V6.1 and PP1S287_51V6.2 (putative 2-ODDs from

751

Physcomitrella patens), and StF3H (Solanum tuberosum). Accession numbers are given in

752

the parentheses.

753 754

Figure 4. Expression, purification, and enzyme activity of recombinant AcFLS proteins. A,

755

SDS-PAGE

756

GST-AcFLS-H6 in E. coli. M, Molecular weight size marker; 1, E. coli lysate before

757

induction; 2, E. coli lysate after induction; 3, soluble fraction isolated after sonication of

758

induced cells; 4, purified recombinant GST-AcFLSs. The molecular weights of the

759

recombinant proteins are calculated as approximately 65 kDa (GST, 27 kDa; AcFLS, 38

760

kDa), and the arrow indicates that the recombinant proteins migrated to the predicted size in

761

SDS-PAGE. B, HPLC chromatograms of products kaempferol (K) and quercetin (Q) from

762

the in vitro reactions of GST-AcFLS-HRB and GST-AcFLS-H6 with dihydrokaempferol

763

(DHK) and dihydroquercetin (DHQ) as substrates. The chromatograms were recorded at

764

370 nm.

analysis of expression and purification of GST-AcFLS-HRB and

765


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766

Figure 5. Gene expression analysis of flavonoid biosynthesis genes in onion. A,

767

Transcription levels of structural genes of the flavonoid biosynthesis pathway in different

768

tissues of HRB and H6 were analyzed by qPCR. Each reaction was normalized to the

769

expression level of Acß-Tubulin gene. Mean values were obtained from three independent

770

replicates. B, The 3-month-old seedlings of H6 and HRB used for total RNA extraction

771

from different tissues.

772 773

Figure 6. UPLC analysis of flavonol and cyanidin aglycones extracted from different

774

tissues of H6 and HRB. A, UPLC chromatograms at 350 nm for analysis of flavonol

775

aglycones in acid hydrolyzed extracts. The retention times and peaks corresponding to each

776

flavonol aglycone (quercetin (Q), kaempferol (K), and isorhamnetin (IR) aglycone) are

777

represented by dashed lines and arrows. B, UPLC chromatograms at 520 nm for analysis of

778

cyanidin aglycones in acid hydrolyzed extracts. C, cyanidin aglycone. C, Contents of Q, K,

779

IR, and C measured from triplicate experiments.

780 781

Figure 7. UPLC analysis of flavonol and cyanidin-glucosides extracted from the sheaths of

782

H6 and HRB. A, UPLC chromatograms at 350 nm and 520 nm for analysis of

783

flavonol-glucosides and cyanidin-glucosides, respectively. Peak 1-9 correspond to

784

quercetin 3,4'-diglucoside, isorhamnetin 3,4'-diglucoside, quercetin 3-glucoside, quercetin

785

4'-glucoside, isorhamnetin 4'-glucoside, quercetin, cyanidin 3-glucoside, cyanidin

786

3-(6''-malonylglucoside), and cyanidin 3-(6''-malony-laminaribioside), respectively. B,



787

Contents of each flavonol and cyanidin-glucosides corresponding to the peak 1-9. F-g,

788

flavonol-glucosides; C-g, cyanidin-glucosides.


Page 36 of 46

Page 37 of 46


Table 1. List of primers used for qPCR Target Accession

Forward (5’ → 3’)

Reverse (5’ → 3’)

gene

number

CHS-A

AY221244.1

ATGGGAATATGTCGAGCGCATG

AACTACTTCACCCTAACCATCAATGGC

CHI

AY700850.1

TGGTTCATCCATTCTCTTCACTCATACC

TCTGACAATCTCGATGCTATGCTTAGC

F3H

AY221246.1

AGGTAATTGTATATCCTCTGGCAATCAGG

CTAGGCTAGTATCTCATCAATGGCCTTG

FLS

AY221247.1

ACACTGACATGTCCAGCCTCACC

TTACCGTTGTTCTGTGTAGCACGC

F3’H

AY541035.1

AGTTGGACATGGAAGAGGCTTATGG

TATGACACGCAACCTCAGATGAGC

DFR

AY221249.1

GCATGAAGAGAATGAACCAATCGC

GGTTTACAGTTCAATTCCTCTGTGCAC

ANS

AY221248.1

TGGATGAGGTTGTGACAGACGATG

TTGATCACCATTACACTGATGATGGATC

TUB

AA451549

TCAGTCCAGTAGGAGGAATGTCGC

CTGTCTTCAGAGGCAAGATGAGCAC

Table 2. Kinetic values of the two recombinant AcFLSs for different substrates Protein

Substrate

Km(μM)

Kcat(s-1)

Kcat/Km(M-1s-1)

GST-AcFLS-H6

DHK

20.319 ± 1.796

0.0014 ± 0.00004

69.276

DHQ

24.425 ± 1.629

0.0096 ± 0.00056

392.357

DHK

15.515 ± 1.377

0.0024 ± 0.00010

153.805

DHQ

26.323 ± 0.179

0.0211 ± 0.00082

800.106

GST-AcFLS-HRB

DHK: dihydrokaempferol; DHQ: dihydroquercetin. The data represent the means ±SD of three independent experiments.



Figure 1


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Figure 3


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Figure 5


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Figure 6



Figure 7


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Graphic for table of contents


Journal of Agricultural and Food Chemistry Page 46 of 46


Comparative Analysis of Two Flavonol Synthases from Different

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