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Reduction of Dihydrokaempferol by Vitis vinfera Dihydroflavonol 4-Reductase to Produce Orange Pelargonidin-type Anthocyanins Sha Xie, Ting Zhao, Zhen-Wen Zhang, and Jiangfei Meng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05766 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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Journal of Agricultural and Food Chemistry

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Reduction of Dihydrokaempferol by Vitis vinfera Dihydroflavonol

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4-Reductase to Produce Orange Pelargonidin-type Anthocyanins

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Sha Xie†, Ting Zhao†, Zhenwen Zhang†,‡* and Jiangfei Meng†*

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†

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‡

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*Corresponding author (Tel: +86-29-8709-1847 (Z.Z.); Fax: +86-29-8709-1099 (Z.Z.); E-mail:

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[email protected] (Z.Z.))

College of Enology, Northwest A & F University, No. 22 Xinong Road, Yangling 712100, China; Shaanxi Engineering Research Center for Viti-Viniculture, Yangling 712100, China;

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ACS Paragon Plus Environment


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ABSTRACT

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Vitis vinifera has been thought to be unable to produce pelargonidin-type anthocyanins because its

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dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. However, in

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this study, pelargonidin 3-O-glucoside was detected in the skin of V. vinifera 'Pinot Noir',

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'Cabernet Sauvignon' and 'Yan73', as well as in the flesh of 'Yan73' using HPLC−ESI-MS/MS.

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Additionally, pelargonidin 3-O-(6-acetyl)-glucoside was detected in 'Yan73' skin and flesh for the

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first time. To further confirm the presence of pelargonidin-type anthocyanins in these grape

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cultivars, their DFRs were cloned, expressed in Escherichia coli and purified. An enzyme activity

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analysis revealed that V. vinifera DFR

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leucopelargonidin, although it preferred dihydroquercetin and dihydromyricetin as substrates.

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Thus, the existence of a pelargonidin-based anthocyanin biosynthetic pathway was confirmed in V.

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vinifera using mass spectrometric and enzymatic methods and redirected anthocyanin biosynthesis

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in V. vinifera L. cultivars.

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KEYWORDS: Pelargonidin-based anthocyanins; Vitis vinifera; Dihydroflavonol 4-reductase

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(DFR); Enzyme activity; HPLC−ESI-MS/MS

can reduce dihydrokaempferol to produce

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INTRODUCTION

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Anthocyanins, a class of flavonoids, confer the orange-to-blue colors in higher plants. The six

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major anthocyanidins in plants are pelargonidin, 8 (Figure 1), cyanidin, 7 (Figure 1), peonidin,

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delphinidin, 9 (Figure 1), petunidin and malvidin.1 The B-ring hydroxylation and methylation of

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anthocyanins promote the development of plant color.2 With the increased numbers of hydroxyl

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groups, the color is bluer. The methylation of anthocyanins leads to a slight reddening.3

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Anthocyanidins are commonly glycosylated at the C3- or the C5-position to produce

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corresponding individual anthocyanins. The glycosylation of anthocyanins causes a slight red shift.

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These glycosyl moieties are further modified by aromatic and/or aliphatic acyl moieties. Aromatic

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acylation results in a blue shift, while aliphatic acylation does not alter the color.3 In general,

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limited plant species can produce all possible colors. This can be the result of the absence or

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mutation of an anthocyanin biosynthetic gene or the substrate specificity of a vital anthocyanin

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biosynthetic enzyme.4 For example, carnation, rose and chrysanthemum cannot form purple

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delphinidin-based anthocyanins owing to the absence of flavonoid 3′,5′-hydroxylase (F3′5′H)

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activity.2,5 Petunia hybrida and Cymbidium hybrida are strikingly devoid of orange

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pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) cannot

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efficiently accept dihydrokaempferol, 2 (Figure 1) as substrate.6 Grape berries are believed to have

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delphinidin- and cyanidin-type anthocyanins but not pelargonidin-type anthocyanins.7,8 However,

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recently pelargonidin 3-O-glucoside was detected in several V. vinifera grapes.9,1 This indicated

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that the V. vinifera DFR could catalyze dihydrokaempferol, 2, to produce pelargonidin-based

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anthocyanins, but no in vitro experimental evidence for this hypothesis exists.

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DFR is an important regulatory point in the anthocyanin biosynthetic pathway, and it controls the 3



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flux into biosynthetic pathway branches leading to distinct anthocyanin profiles.6 With NADPH as

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a cofactor, DFR catalyzes the reduction of dihydroquercetin, 1 (Figure 1), dihydrokaempferol, 2,

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and dihydromyricetin, 3 (Figure 1) to their respective leucoanthocyanidins. They are subsequently

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converted to cyanidin, 7, pelargonidin, 8, and delphinidin, 9, respectively (Figure 1).10 DFR has

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different substrate specificities in different species, and amino acid residues 134 and 145 play

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important roles in the substrate specificity.10,11 The substrate specificities of DFRs have been

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studied extensively in lots of plants,12,11,13,14but little is known in grape berries. The discovery of

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pelargonidin-type anthocyanins in grape berries provided new insights into the anthocyanin

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biosynthesis pathway in V. vinifera. Grape DFR activity is very essential for fully interpreting

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anthocyanin biosynthesis.

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In this study, we analyzed the anthocyanin profiles of Vitis vinifera L. cv. Pinot Noir, Cabernet

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Sauvignon and Yan73 using HPLC−ESI-MS/MS. 'Yan73', a Chinses teinturier cultivar, is a cross

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between 'Alicante Bouschet' (V. vinifera L.) and 'Muscat Hamburg' (V. vinifera L.). It can

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synthesize anthocyanins in the skin, pulp, rachis and pedicels, and is commonly used to blend with

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pale red wines to enhance their color.15 Consistent with previous reports,9,1 pelargonidin-type

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anthocyanins were detected in the skin of 'Pinot Noir', 'Cabernet Sauvignon' and 'Yan73', as well

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as the flesh of 'Yan73'. To further confirm this finding, the cDNAs encoding DFRs were cloned

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from these V. vinifera L. cultivars, and the DFR proteins were expressed in an Escherichia coli

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system. DFR proteins were purified using a nickel-affinity column, and their enzyme activities

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were determined. This study brings a new understanding of the V. vinifera anthocyanin

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biosynthetic pathway.

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MATERIALS AND METHODS 4


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Plant Material

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V. vinifera 'Pinot Noir', 'Cabernet Sauvignon' and 'Yan73' berries were sampled from Chateau

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Changyu Verna, Shaanxi, China (108°73 N; 34°33E) at commercial harvest. All of the cultivars

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had been planted since 2009.

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Extraction and Analysis of Anthocyanins of Grape Berries

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Anthocyanins were extracted from grape berries according to the method of Xie et al.16 Three

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independent extractions were performed for each sample. The extracts were stored at -40 °C and

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filtered through 0.45 µm cellulose acetate and nitrocellulose filters (Millipore Co., MA) before

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injection.

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The individual anthocyanins were analyzed using HPLC−ESI-MS/MS with a 1100 series

99

LC-MSD trap VL (Agilent, Santa Clara, CA) equipped with a G1379A degasser, G1312BA

100

quaternary pump, G1313A ALS autosampler, G1316A column compartment, G1315A diode array

101

detector (DAD), and a Kromasil C18 column (250 × 4.6 mm, 5 µm) following our previous

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work.16 All of the samples were analyzed in duplicate.

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Anthocyanins were identified by comparing their elution orders, retention times, and molecular

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and fragment ions with those of the standards and references.17,18 The quantification of

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anthocyanins followed the external-standard method. The anthocyanin content was expressed in

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equivalents of malvidin 3-O-glucoside.

107

Cloning of DFR Candidate Genes

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Total RNA was extracted from the selected grape varieties and the respective cDNAs were

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synthesized using a previously described method.16

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The full-length coding sequences of the DFR were PCR amplified from a cDNA library of the 5



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three V. vinifera cultivars. The primers were designed based on the DFR sequences

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(NM_001281215) published in NCBI. PCR was carried out using the oligonucleotide primers

113

DFR-F

114

(5'-GGTCTTGCCATCTACAGGTTTCTC-3'). The PCR reaction was conducted using Phanta

115

Super-Fidelity DNA polymerase (Vazyme Biotech, Nanjing, China). The PCR product (~1,014 bp)

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was inserted into PMD19-T Easy vector (Takara, Japan) and sequenced.

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Sequence Alignment and Phylogenetic analysis

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The alignment of sequences was performed using DNAMAN 6.0 software. A multiple sequence

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alignment was carried out using MEGA5.1.19 The phylogenetic tree was constructed using the

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neighbor-joining method with MEGA5.1 under default parameters and 1,000 bootstrap

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replicates.19

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Expression and Purification of DFR Proteins

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After confirmation by sequencing, the VvDFR coding region and short poly(A) tail were digested

124

from PMD19-T Easy by BamHI and XhoI and inserted into pET-32a expression vector (Novagen,

125

Madison, WI) to produce pET-32a-VvDFR. The recombinant plasmids were transformed into E.

126

coli strain BL21 (DE3). The plasmid-containing strain was inoculated at 37 °C in Luria-Bertani

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medium containing 100 µg/mL ampicillin at 260 rpm until the OD600 reached 0.6. Then, isopropyl

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β-D-thiogalactoside was added to 0.5 mM final concentration to initiate overexpression, and the

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cells were further cultured at 16 °C, 260 rpm for 24 h. After centrifugation the cell, the supernatant

130

was discarded and the pellet was resuspended in lysis buffer (45 mM imidazole, 20 mM sodium

131

phosphate and 500 mM NaCl, pH 7.4). Cells were sonicated for 5 min (Branson Digital Sonifier).

132

After centrifugation at 10,000 r/min for 20 min at 4 °C, the supernatants were collected.

(5'-ATGGGTTCACAAAGTGAAACCG-3')

6


and

DFR-R

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The purification procedures were performed using a His-GraviTrap nickel-affinity column (GE

134

Healthcare, Chalfont St Giles, Buckinghamshire). After equilibrating the column with 10 mL

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binding buffer (45 mM imidazole, 20 mM sodium phosphate and 500 mM NaCl, pH 7.4), the

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samples were added. Then, the column was eluted with 10 mL binding buffer and 3 mL elution

137

buffer (500 mM imidazole, 20 mM sodium phosphate, 500 mM NaCl, pH 7.4) in turn, and the

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eluate was collected. All steps were performed at 4 °C. The purified proteins were analyzed by

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SDS-PAGE and Coomassie Brilliant Blue R250 staining. Control experiments were conducted

140

with E. coli BL21 (DE3) cells harboring the empty pET-32a vector.

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Enzyme Assays and HPLC−ESI-MS/MS Analysis of DFR reaction products

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Three kinds of dihydroflavonols with different hydroxylation patterns, 1-3, were independently

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used as substrates of DFR. The reaction mixture (3 mL) included 2 mmol/L NADPH, 0.1 mol/L

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potassium phosphate buffer (pH 6.5), 0.1 mmol/L substrate, 1-3, and the purified protein (0.5 mL).

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The reaction was conducted at 37 °C for 1 h. Because leucoanthocyanidin is unstable, an equal

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volume of n-BuOH:HCl, 95:5 (v/v) was added, and the reaction was conducted at 95 °C for 1 h to

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form the anthocyanidins.20,21 The N-BuOH layer was collected and evaporated to dryness in a

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lyophilizer at 30 °C under vacuum. The residue was suspended in 0.25 mL methanol. The reaction

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products were filtered through 0.22 µm cellulose acetate/nitrocellulose filters before injection. All

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flavonoid standards were from Sigma-Aldrich Co. (St. Louis, MO).

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HPLC-DAD and HPLC–PDA–MS/MS–ESI were used in the identification and quantitation of

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DFR reaction products. The HPLC-DAD system included a GT-154 vacuum degasser, two

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LC-10AT pumps, a SIL-10A automatic injector, a CTO-10A column oven, a SPD-M10AVP DAD,

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and a SCL-10AVP system controller (Shimadzu, Kyoto, Japan). A Phenomenex Prodigy RP-18 7



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ODS (3) column (250 × 4.6 mm, 5 µm, Torrance, CA) combined with a Phenomenex Prodigy

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guard column (30 × 4.6 mm, 5 µm) was used. The mobile phases were (A) 5% formic acid (v/v),

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and (B) acetonitrile/methanol (3:7, v/v). The gradient program was 0–30 min with 5-25% solvent

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B, 31–60 min with 25-40% B, 61–70 min with 40-80% B and 71–90 min with 5-25% solvent B.

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The flow rate was 0.8 mL/min. The injection volume was 10 µL. The wavelength scanning range

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of the DADs was 200 nm–600 nm. The quantitative detection wavelength was 520 nm.

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The HPLC–ESI-MS/MS analysis was carried out on an Acquity ultra-performance LC system

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equipped with a Waters Quattro Premier MS (Waters Corp., Milford, MA) coupled with an

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ion-spray interface. The HPLC columns, gradient program, flow rate and injection volume were

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the same as in the HPLC-DAD analysis. The scanning ranges were from m/z 200-900 and from

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m/z 900-2,000.

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Statistical Analysis

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Statistic analysis was performed using OriginPro 8.5 (OriginLab Corporation, Northampton, MA).

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RESULTS AND DISCUSSION

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Mass Spectrometric Evidence Confirming the Presence of Pelargonidin-type

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Anthocyanins

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A total of 14, 17, 19 and 20 anthocyanins were detected in the skin of 'Pinot Noir', 'Cabernet

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Sauvignon' and 'Yan73' as well as the flesh of 'Yan73', respectively (Figure 2A). The ESI-MS

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spectra revealed that a molecular ion at m/z 433 and a fragment ion at m/z 271 were associated

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with peak 6 of 'Pinot Noir', 'Cabernet Sauvignon' and the skin and flesh of 'Yan73' (Figure 2B).

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The m/z 271 fragment ion corresponded to the pelargonidin aglycone,9,22 and the difference

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between the molecular ion and the aglycone ion appeared at m/z 271 (433–162). This indicated a 8


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hexose, which in grape would mostly be glucose (Figure 2C). This anthocyanin was identified as

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pelargonidin 3-O-glucoside. Therefore, pelargonidin 3-O-glucosides were detected in the skin of

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'Pinot Noir', 'Cabernet Sauvignon' and 'Yan73', as well as the flesh of 'Yan73', consistent with

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previous reports.1,15 Peak 12 of 'Yan73' had a molecular ion at m/z 475 and fragment ions at m/z

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271 (Figure 2B), indicating that pelargonidin, acetyl, and hexose groups occur in the structure

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(Figure 2C). Based on previous references,9,23 peak 12 of 'Yan73' was assigned as pelargonidin

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3-O-(6-acetyl)-glucoside. To the best of our knowledge, this is first time that pelargonidin

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3-O-(6-acetyl)-glucoside has been found in the skin and flesh of 'Yan73', although other teinturier

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grape

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3-O-(6-acetyl)-glucoside.9 Pelargonidin 3-O-(6-acetyl)-glucoside was not detected in the skin of

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'Pinot Noir' or 'Cabernet Sauvignon', which was in agreement with a previous report.1 In

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conclusion, pelargonidin-based anthocyanins are actually not absent and could be detected with an

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improved HPLC-DAD-MS/MS analysis in V. vinifera berries.

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Anthocyanin Profiling of Three Grape Cultivars

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Anthocyanins

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pelargonidin-based anthocyanins according to the number of hydroxyl groups on the B-ring. The

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3',5'-di-OH anthocyanins with hydroxylation of the 3',5'-positions of the flavonoid B-ring, contain

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delphinidin-type anthocyanins (blue to purple). The 3′-OH anthocyanins with hydroxylation at the

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3′ positions of the B-ring, include cyanidin-type anthocyanins (red). Pelargonidin-based

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anthocyanins (orange) have no hydroxyl group at the 3′ and/or 5′ position of the B-ring. Flavonoid

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3-hydroxylase (F3′H), F3′5′H and DFR are three key enzymes controlling the flux into

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biosynthetic pathway branches that lead to the production of 3',5'-di-OH anthocyanins, 3′-OH

cultivars,

are

such

as

divided

V.

into

vinifera

Alicante

3',5'-di-OH

Bouschet

anthocyanins,

9


contained

3'-OH

pelargonidin

anthocyanins

and


199

anthocyanins and pelargonidin-based anthocyanins, respectively (Figure 1). 'Yan73' skin contained

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the highest total concentration of anthocyanins among the three cultivars, followed by 'Yan73'

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flesh, while 'Pinot Noir' skin had the lowest total content (Figure 3). The 3',5'-di-OH anthocyanins

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and 3′-OH anthocyanins were the predominant anthocyanins, jointly accounting for 99.6-100% of

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total anthocyanins (Figure 3). This indicated the high activity levels of F3′H and F3′5′H.

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Pelargonidin-based anthocyanins were present in trace levels in the skin of 'Pinot Noir' and

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'Cabernet Sauvignon', but the 'Yan73' skin and flesh contained relatively higher concentrations.

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This may be because the F3′5′H and F3′H enzymes compete for substrates with the DFR enzyme

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(Figure 1), and most of the dihydrokaempferol, 2, was used to form 3',5'-di-OH and 3′-OH

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anthocyanins but not to produce pelargonidin-based anthocyanins in grapes.1 Additionally,

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pelargonidin 3-O-(6-acetyl)-glucoside was detected in 'Yan73' skin and flesh for the first time. The

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percentages of 3',5'-di-OH anthocyanins in the skin of the three cultivars were greater than the

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corresponding 3′-OH anthocyanins, suggesting that F3′5′H activity prevails over F3′H in the skin

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of these grape cultivars. Similar results were found in the previous studies of these grape

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cultivars.7,24,15 However, the flesh of 'Yan73' contained similar levels of 3',5'-di-OH and 3′-OH

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anthocyanins, which was consistent with studies of another teinturier cultivar.25 Thus, the flux

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down the trihydroxylated branch of the pathway is proportionally less in 'Yan73' flesh than in its

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skin, which might result from the different activity levels of F3′H, F3′5′H and DFR enzymes

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toward dihydrokaempferol, 2, in 'Yan73' skin and flesh tissues.9,25

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Sequence and Phylogenetic Analyses

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DFR is an important regulatory point in controlling the flux into the anthocyanin biosynthetic

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pathway branches that lead to distinct anthocyanin profiles. Petunia and Cymbidium cannot 10


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generate pelargonidin-type orange flower colors even when both F3′H and F3′5′H are absent

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because DFRs from these species cannot reduce dihydrokaempferol, 2, efficiently.13,4 To further

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confirm the presence of pelargonidin-base anthocyanins in our grape cultivars, the full-length

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DFR cDNAs of 1,014 bp from these grapes were obtained and their sequences were analyzed. The

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VvDFR cDNA contained 1,034 nucleotides, encoding 336 amino acids. The similarity level of the

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nucleotide sequences of the cloned DFR genes from these grape cultivars was 99.95%, but that of

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putative amino acid sequences was 100%. A multiple sequence alignment showed that DFR

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proteins contained two conserved domains: NADP-binding and putative substrate-binding regions

229

(Figure 4A), which was consistent with a previous report.26 Amino acid residue 134 plays an

230

important role in substrate specificity with DFRs being divided into asparagine (Asn)-type DFRs,

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aspartic acid (Asp)-type DFRs and non-Asn/Asp-type DFRs.10,11 Asn-type DFRs are widely

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distributed in plants, including the three grape cultivars we selected, whereas Asp-type DFRs are

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mainly found in the eudicots, such as Petunia, Lycopersicon esculentum, Calibrachoa and

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Solanum tuberosum. Asn-type DFRs can utilize all three dihydroflavonols, 1-3, as substrates,

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while Asp-type DFRs cannot catalyze dihydrokaempferol, 2, efficiently.4,27 However, a recent

236

study showed that no pelargonidin, 8, was detected in flowers of Freesia hybrida although their

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DFR belongs to the Asn-type.26 In our study, VvDFR belonged to the Asn-type, and we detected

238

the presence of pelargonidin-base anthocyanins. Residues 145 in the binding domain also affects

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the substrate specificity of the DFR enzyme.13 A glutamic acid at this residue is conserved in most

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DFRs, including that of our grape cultivars, while some DFRs have glutamine (Petunia and

241

Hordeum vulgare) or histidine (Oryza sativa and Zea mays).

242

The phylogenetic tree showed a clear segregation of DFR proteins into monocotyledons and 11



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dicotyledons (Figure 4B), which indicated that the DFR divergence occurred after the divergence

244

of monocotyledons and dicotyledons.28 The sequence analysis showed that VvDFR is most closely

245

related to DFRs from Malus domestica and Crataegus monogyna (79.02% and 78.96% amino acid

246

homology levels, respectively). A previous study reported that Malus domestica DFR accepts

247

dihydroquercetin, 1, and dihydrokaempferol, 2, equally as substrates.12 VvDFR, MdDFR and

248

CmDFR in our study all belonged to the Asn-type of DFRs. Asn-type DFRs can utilize all three

249

dihydroflavonols, 1-3, as substrates. CaDFR, LeDFR, PeDFR and StDFR, which are grouped into

250

the Asp-type of DFRs, showed a distant relationship to VvDFR in the phylogenetic tree. Asp-type

251

DFRs may not efficiently catalyze dihydrokaempferol, 2, to produce pelargonidin-based

252

anthocyanins,4,26 while pelargonidin, 8, was detected in the Calibrachoa hybrid that harbored a

253

DFR gene belonging to the Asp-type.10 This may be because amino acid residue 134 of the DFR is

254

not solely responsible for its activity toward dihydroflavonols.20 Domain swapping experiments

255

between Petunia and Gerbera have shown that the ability of DFR to metabolize

256

dihydrokaempferol, 2, is encoded in the first 170 amino acids.13 Further studies with site-directed

257

mutagenesis are needed to find out the influence of specific amino acid residues on the activity

258

levels and substrate preferences of DFRs.

259

Enzyme Assay and HPLC−ESI-MS/MS Analysis of DFR Reaction Products

260

An SDS-PAGE of the expressed DFR proteins showed bands near the predicted molecular mass

261

of 37.6 kDa (Figure 5), which was consistent with a previous report.14 Our grape DFR activity

262

levels versus the three dihydroflavonols, 1-3, were determined using HPLC−ESI-MS/MS.

263

Because of instability of leucoanthocyanidins, the natural DFR products produced by reducing

264

dihydroflavonols, the reaction was chemically performed by acidic alcohols to generate the 12


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corresponding anthocyanidins.20 Figure 6A shows the HPLC chromatogram and mass spectra of

266

the delphinidin, cyanidin and pelargonidin standards. When dihydromyricetin, 3, was used as the

267

substrate, peak 1 was produced and the corresponding compounds displayed a fuchsia coloration

268

(Figure 6B). Peak 1 eluted at the same retention time, gave the same color and mass spectrum

269

with major fragments at m/z 303,257 and 229 as the delphinidin standard (Figure 6A). The

270

fragment mass at m/z 303 is equal to the mass of delphinidin, 9.29 Thus, peak 1 was assigned as

271

delphinidin, 9. These results indicated that VvDFR can convert dihydromyricetin, 3, to

272

leucodelphinidin, 6 (Figure 1). When dihydroquercetin, 1, was used as the substrate, peak 2 was

273

produced, and the corresponding compounds displayed a red coloration (Figure 6B). Peak 2

274

matched the retention time, color and mass spectrum of the cyanidin-standard (Figure 6A). They

275

both had molecular ions at m/z 287 and two fragment ions at m/z 213 and m/z 137, which was

276

consistent with the previously reported mass spectrum of cyanidin, 7.29,6 Therefore, Peak 2 was

277

identified as cyanidin, 7, indicating that VvDFR can efficiently catalyze the reduction of

278

dihydroquercetin, 1, to leucocyanidin, 4 (Figure 1). When dihydrokaempferol, 2, was used as the

279

substrate, peak 3 was produced, and the corresponding compounds displayed a peach coloration

280

(Figure 6B). Peak 3 was identified as pelargonidin, 8, by comparing its retention time, color and

281

MS data (m/z 271,121 and 141) with the pelargonidin standard (Figure 6A) and the reference,29

282

which suggested that VvDFR can reduce dihydrokaempferol, 2, to produce leucopelargonidin, 5

283

(Figure 1). Thus, VvDFR could utilize all three dihydroflavonols, 1-3, as substrates. DFR

284

enzymatic reactions versus the three dihydroflavonols, 1-3, were conducted under the same

285

conditions. The quantitation analysis of reaction products showed that VvDFR preferred

286

dihydroquercetin, 1, over dihydromyricetin, 3, and only converted dihydrokaempferol, 2, to a 13



287

minor extent with cyanidin, 7, delphinidin, 9, and pelargonidin, 8, having the concentrations of

288

190.97 mg/L, 66.67 mg/L and 10.21 mg/L, respectively. However, based on the anthocyanin

289

profiling of grape berries mentioned above, delphinidin-based anthocyanins were more abundant

290

than cyanidin-based anthocyanins, suggesting that more dihydromyricetin, 3, was used to form

291

delphinidin-based anthocyanins. This may be because the F3′5′H activity was higher than the

292

F3′H activity in grape berries, resulting in a greater production of dihydromyricetin, 3, indicating

293

that anthocyanin profiling in grape berries is a result of the cooperation of many related genes. In

294

summary, our mass spectrometric and enzymatic evidence confirmed the existence of

295

pelargonidin-based anthocyanins, which indicated that the pelargonidin-based anthocyanin

296

biosynthetic pathway exists in V. vinifera. Pelargonidin-based anthocyanins were detected at

297

trace levels in V. vinifera. It is possible that VvDFR had a higher affinity for dihydroquercetin, 1,

298

and dihydromyricetin, 3, producing more cyanidin-based and delphinidin-based anthocyanins,

299

and, therefore, less dihydrokaempferol, 2, was used to form pelargonidin-based anthocyanins.

300

In conclusion, our mass spectrometric and enzymatic methods confirmed the existence of a

301

pelargonidin-based anthocyanin biosynthetic pathway in V. vinifera. Additionally, pelargonidin

302

3-O-(6-acetyl)-glucoside was detected in the skin and flesh of 'Yan73' for the first time. This

303

finding results in a new understanding of the anthocyanin biosynthetic pathway in V. vinifera.

304

Funding sources

305

This work was supported by the China Agriculture Research System for Grape Industry

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(CARS-29-zp-6).

307

Acknowledgements

308

We are grateful to the Center for Viticulture and Enology, China Agriculture University for access 14


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to the HPLC−ESI-MS/MS equipment.

310

Supporting Information

311

The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

312

Notes

313

The authors declare no conflict of interest.

314

References

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419

Figure Captions

420

Figure 1. Anthocyanidin biosynthetic pathways. 1, dihydroquercetin; 2, dihydrokaempferol; 3,

421

dihydromyricetin; 4, leucocyanidin; 5, leucopelargonidin; 6, leucodelphinidin; 7, cyanidin; 8,

422

pelargonidin; 9, delphinidin; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase;

423

DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase.

424

Figure 2. Detection of pelargonidin 3-O-glucoside and its derivatives in three V. vinifera cultivars.

425

A) Reversed-phase HPLC chromatograms of anthocyanins from the three grape cultivars. B) Ion

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mass spectra of pelargonidin 3-O-glucoside and pelargonidin-3-O-(6-acetyl)-glucoside detected in

427

three grape cultivars. The peak numbers correspond to Figure 2A. C) Mechanism to produce the

428

main fragment ion of pelargonidin, 8, from the cation of pelargonidin 3-O-glucoside and

429

pelargonidin 3-O-(6-acetyl)-glucoside.

430

Figure 3. Anthocyanin profiling of three grape cultivars. PN, Pinot Noir; CS, Cabernet Sauvignon;

431

YS, skin of Yan73; YF, flesh of Yan73; “Trace” indicates that trace levels of pelargonidin-based

432

anthocyanins were detected in PN and CS.

433

Figure 4. Alignment of amino acid sequences and phylogenetic analysis of DFR proteins in the

434

three Vitis vinifera cultivars with proteins from other species. A) Alignment of the amino acid

435

sequences of the DFR proteins in the three V. vinifera cultivars with proteins from other species.

436

The putative NADP-binding site and substrate-binding region are boxed in green. B) Phylogenetic

437

analysis of the DFR proteins in the three V. vinifera cultivars with proteins from other species.

438

Numbers indicate bootstrap values for 1,000 replicates. V. vinifera proteins are indicated with

439

green circles. PN, Pinot Noir; CS, Cabernet Sauvignon; YS, skin of Yan73; YF, flesh of Yan73.

440

The GenBank accession numbers of the DFR protein sequences are as follows: Agapanthus 20


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praecox ApDFR (BAE78769); Allium cepa AcDFR (AAO63026); Antirrhinum majus AmDFR

442

(X15536); Arabidopsis thaliana AtDFR (NM_123645); Bromheadia finlaysoniana BfDFR

443

(AF007096); Calibrachoa hybrid CaDFR (KC140106); Callistephus chinensis CcDFR (Z67981);

444

Chrysanthemum ChDFR1 (EF094935); Chrysanthemum ChDFR2 (EF094936); Crataegus

445

monogyna CmDFR (AAX16491); Cymbidium CyDFR (AF017451); Dianthus caryophyllus

446

DcDFR1 (AB071787); Dianthus caryophyllus DcDFR2 (Z67983); Fragaria× ananassa FaDFR

447

(AAX12421); Gerbera jamesonii GjDFR (KF734593); Hordeum vulgare HvDFR (S69616);

448

Lycopersicon esculentum LeDFR (Z18277); Malus domestica MdDFR (AAO39816); Oryza sativa

449

OsDFR (AB003495); Paeonia lactiflora PlDFR (JQ070804); Petunia × hybrid PeDFR

450

(AF233639); Rosa rugose RrDFR (KT809350); Zea mays ZmDFR1 (Y16040); Zea mays

451

ZmDFR2 (X05068); Solanum tuberosum StDFR (AFQ98276).

452

Figure 5. SDS-PAGE analysis of His-tagged DFR protein. Lane 1, Total protein of E. coli BL21

453

(DE3) harboring the empty pET-32a expression vector; Lane 2, Total protein of E. coli BL21

454

(DE3) containing the expression plasmid pET-32a-VvDFR; Lane 3, His-tagged DFR protein

455

purified using a nickel-affinity column.

456

Figure 6. Enzyme assay of His-tagged DFR protein. A) HPLC-MS/MS analysis of the standards

457

of delphinidin, 9, cyanidin, 7, and pelargonidin, 8. The concentrations of these standards are 25

458

mg/L. B) HPLC-MS/MS analysis of DFR reaction products. The concentrations of Peak 1, Peak 2

459

and Peak 3 are 66.67 mg/L, 190.97 mg/L and 10.21 mg/L, respectively. Numbers in brackets are

460

retention times. Numbers in the green boxes are the major fragment masses.

461

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