Root Glucosinolate Profiles for Screening of Radish (Raphanus

Dec 17, 2015 - Radish (Raphanus sativus L.), a root vegetable, is rich in glucosinolates (GLs), which are beneficial secondary metabolites for human h...
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Root Glucosinolate Profiles for Screening of Radish (Raphanus sativus L.) Genetic Resources Gibum Yi,*,†,‡,⊥ Sooyeon Lim,†,§,⊥ Won Byoung Chae,∥ Jeong Eun Park,† Hye Rang Park,† Eun Jin Lee,†,§ and Jin Hoe Huh*,†,‡,§ †

Department of Plant Science, ‡Plant Genomics and Breeding Institute, §Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea ∥ Department of Horticultural Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun, Jeollabuk-do 55365, Korea S Supporting Information *

ABSTRACT: Radish (Raphanus sativus L.), a root vegetable, is rich in glucosinolates (GLs), which are beneficial secondary metabolites for human health. To investigate the genetic variations in GL content in radish roots and the relationship with other root phenotypes, we analyzed 71 accessions from 23 different countries for GLs using HPLC. The most abundant GL in radish roots was glucoraphasatin, a GL with four-carbon aliphatic side chain. The content of glucoraphasatin represented at least 84.5% of the total GL content. Indolyl GL represented only 3.1% of the total GL at its maximum. The principal component analysis of GL profiles with various root phenotypes showed that four different genotypes exist in the 71 accessions. Although no strong correlation with GL content and root phenotype was observed, the varied GL content levels demonstrate the genetic diversity of GL content, and the amount that GLs could be potentially improved by breeding in radishes. KEYWORDS: glucosinolates, radish, glucoraphasatin, Raphanus sativus, secondary metabolite

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The fluctuation of GL content is under the effect of both environmental factors, such as climate and cultivation condition, and development stages. However, genetic variation is one of the major factors that determines GL content.8 Therefore, GL composition and content are important breeding targets. Large variations in genetic resources have been screened in Brassica vegetables such as oilseed rape, broccoli, turnip, and pak choi.9−12 A high GL-containing broccoli (B. oleracea) cultivar, “Beneforte”, was successfully bred and commercialized.13 However, GL content and composition in radish have only been investigated in a few studies.14−17 In addition, there is a study that analyzed GL content variation in wild radish; however, it mainly focused on its biofumigation potential.16 Here, we analyzed GL profiles in root samples and root phenotypes of 71 radish accessions to investigate the genetic variations and the relationship between GL profiles and root phenotypes.

INTRODUCTION Glucosinolates (GLs) are sulfur-containing phytochemical compounds that are widely found in the order Brassicale. GLs are enzymatically hydrolyzed to isothiocyanates (ITCs), thiocyanates, nitriles, etc. by myrosinases.1 GLs and one of their derivatives, ITCs, are beneficial to human health because of their effects on carcinogenesis, inflammation, cardiovascular protection, etc.2 There is increasing interest surrounding GLs and the need for vegetables as a source of GL. Over 200 different GLs have been reported, and each vegetable species has a specific profile.3 For example, Brassica rapa is rich in aliphatic GLs with 4C and 5C side-chains, whereas B. oleracea is rich in homomethionine-derived aliphatic GLs with a 3C sidechain (Figure 1). Radish (Raphanus sativus L.), a root vegetable and a member of the Brassicaceae family, has a specific GL profile that is abundant in glucoraphasatin (GRH).1 It is also known to have a higher converting ratio to produce ITCs from GLs compared to broccoli.4 ITCs produced by the hydrolysis of GLs are known to be the major pungent compounds that are responsible for the unique taste in radish, which is mostly caused by the production of raphasatin from GRH.5 Raphasatin was reported to have an antitumorigenic effect promoting anticancer activity when it is combined with other phytochemicals.6,7 The large amount of GLs and a high converting ratio make radish a preferable source of GLs for the human diet. © XXXX American Chemical Society

Received: September 18, 2015 Revised: December 11, 2015 Accepted: December 17, 2015

A

DOI: 10.1021/acs.jafc.5b04575 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Schematic pathway of GL biosynthesis. The GLs analyzed in this study indicated as abbreviations. Aliphatic GLs biosynthesis pathway was modified from Ishida et al.15and indoyl GLs pathway was adopted from Pfalz et al.27 Dotted line indicates lack of experimental evidence.

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GL was individually extracted and analyzed. Cuboidal pieces (1 cm3) of root tissue were collected from a horizontal, 1 cm thick disc in the middle of the root. The root samples were kept at −80 °C and freezedried in the order sample collection occurred. The lyophilized root samples were kept in plastic bags with silica gel in the dark until use. Then, the samples were ground using a food blender (BL1201KR, Tefal, France) at a low speed setting for 30 s. The ground samples (200 mg) were combined with 10 mL of boiled 80% methanol and incubated for 30 min at 90 °C.19 The methanol extracts were evaporated under N2 gas and resolved in 3 mL of 80% methanol. The extracts were loaded to the DEAE sephadex-A25 column and washed twice with 1 mL of 20 mM sodium acetate (pH 5.0). The purified sulfatase (75 μL, 150 U mL−1) was added on the top of the column and incubated for 16 h at 35 °C. Desulfo-GLs were eluted three times with 0.5 mL HPLC-grade water. Eluted solutions were combined and concentrated to 0.5 mL for HPLC analysis and LC−MS/MS. Each sample was filtered through a 0.45 μm PVDF membrane syringe filter before injection. The GL content was calculated as microgram per gram of dry weight (μg g−1 DW) and further converted to nmol g−1 DW. HPLC Analysis. HPLC was used for the determination of desulfoGLs and was performed using an LC system (YL9111, Younglin Instrument, Korea) equipped with a PDA detector (YL9160, Younglin Instrument, Korea). An Eclipse Plus C-18 column (4.6 × 250 mm, Agilent Co., U.S.A.) was used with incubation at 35 °C. A gradient mobile phase of water (A) and acetonitrile (B, Burdick and Jackson, U.S.A.) separated the compounds by increasing B from 2% to 20% in 25 min and switching to an additional linear gradient of 20% to 100% B for the next 35 min. For desulfo-GL quantification, sinigrin monohydrate (Sigma-Aldrich, U.S.A.) was used as an external standard. The absorbance at 229 nm was monitored for various concentrations (0.0−0.125 mg mL−1) of desulfo-sinigrin, and each GL content was calculated based on the UV response factors of other GLs relative to sinigrin.20 Identification of Individual GLs. GLs in radish roots were identified as desulfo-GL structures using a Q Exactive Hybrid Quadrupole-Orbitrap instrument equipped with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific Co., U.S.A.). HPLC chromatography was performed at a 150 μL min−1 flow rate, with a 10 μL injection volume and a 35 °C column temperature equipped with an INNO 10 column (2.0 × 100 mm2, Young Jin Biochrom,

MATERIALS AND METHODS

Plant Materials and Cultivation. A total of 71 radish (R. sativus L.) accessions were used for this study, including 40 accessions from RDA-GENEBANK (www.genebank.go.kr) collections and 31 cultivars and inbred lines from the National Institute of Horticultural and Herbal Science (NIHHS). In addition, two accessions of turnip (B. rapa) were also analyzed (also available at RDA-GENEBANK). All accessions were grown with conventional culture methods in the NIHHS field in Suwon, Korea. The field was prepared with preplant broadcast manure at a dose of 10 000 kg ha−1 and basal fertilizer containing 160 kg ha−1 N, 80 kg ha−1 K2O, and 160 kg ha−1 P2O5. Twenty seeds per accession were sown on September 3, 2013 with 25 cm spacing within-row and 35 cm spacing between rows. During the growth time the average of lower and higher temperature was 12.3 to 22.5 °C and 70% of relative humidity on average. The morphological and agronomical traits of roots were investigated at 70 days after planting. Morphological traits such as root shape and exterior color were investigated based on “IBPGR descriptors for Brassica and Raphanus, 1990”. Root length was measured for three roots per accession, and root pithiness was discerned by visual comparison of the longitudinal cut of roots using a scale ranging from 0 (no pithiness) to 9 (highest pithiness). The sugar content of roots was measured using a portable refractometer (PAL-1, Atago, Inc., Japan). Preparation of Sulfatase and a DEAE Column. Sulfatase from Helix pomatia was purchased from Sigma-Aldrich Chemical (U.S.A.). Sulfatase (10 000 U g−1) was purified by ethanol precipitation.18 In summary, the 75 mg sulfatase powder was combined with 6 mL of cold distilled water and vortexed. The solution was combined with 4 mL of cold ethanol and directly centrifuged at 3000g for 10 min at 4 °C. The supernatant was transferred to a 50 mL conical tube and mixed with 20 mL of cold ethanol and centrifuged again (at 3000g, 4 °C for 6 min). The pellet was collected and evaporated for several minutes to remove the remaining ethanol. Purified sulfatase was dissolved in cold water (150 U mL−1), aliquoted into microtubes, and stored at −20 °C until use. In total, 100 mg of DEAE sephadex-A25 resin (GE Healthcare, Sweden) was activated using 2 M acetic acid overnight in a microtube. Activated resin was packed in a 5 mL disposable poly prep column (Thermo Scientific Co., U.S.A.). Before loading a sample, the column was rinsed twice with distilled water. Extraction of GLs. Three individual roots per accession were used for biological replicates. They were sampled at 70 days after planting, B

DOI: 10.1021/acs.jafc.5b04575 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Glucosinolates Detected in Radish Roots common name aliphatic Progoitrin Glucoraphenin Gluconapin Glucobrassicanapin Glucoerucin Glucoraphasatin indolyl 4-Hydroxyglucobrassicin 4-Methoxyglucobrassicin

side chain

molecular formula

Mw

desulfo-Mw

RT (min)

PRO GRE GNP GBN GER GRH

2-Hydroxy-3-butenyl 4-Methylsulfinyl-3-butenyl 3-Butenyl Pent-4-enyl 4-Methylthiobutyl 4-Methylthio-3-butenyl

C11H19NO10S2 C12H21NO10S3 C11H19NO9S2 C12H21NO9S2 C12H23NO9S3 C12H21NO9S3

389 435 373 387 421 419

309 355 293 307 341 339

6.8 8.2 9.3 15.9 21.0 21.9

4HGBS 4MGBS

4-Hydroxy-3-indolylmethyl 4-Methoxy-3-indolylmethyl

C16H20N2O10S2 C17H22N2O10S2

464 478

384 398

23.5 26.8

abbreviation

Figure 2. HPLC chromatograms of individual desulfo-glucosinolates in accession G064, G187, and G032, representative samples for each PCA group. Peak identifications: Glucoraphenin (GRE), Gluconapin (GNP), Glucobrassicanapin (GBN), Glucoerucin (GER), Glucoraphasatin (GRH), 4-Hydroxyglucobrassicin (4HGBS), and 4-Methoxyglucobrassicin (4MGBS). Korea). The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B). The initial composition of the mobile phase consisted of 90% A. The A decreased from 90% to 20% in 14 min and was maintained for the next 15 min for washing. The portion of solution A was switched to an additional linear gradient of 20% to 90% B for up to 16 min and equilibrated for 20 min. Desulfo-GLs were ionized using electrospray ionization (ESI) in the capillary column. The temperature of the capillary column was maintained at 320 °C, and the voltage was set to 3.5 kV. The N2 gas flow was 12 L min−1, and the nebulizer pressure was 35 psi. The average scan time was 0.01 min, and the average time to change polarity was 0.02 min. The system was operated in both negative and positive modes and scanned from m/z 50 to 1000. After injection into the UPLC−ESI−MS/MS system,

additional confirmation on the identified desulfo-GLs was carried out by performing a selective ion scanning and matching with mass fragments of reference information.21,22 The data set for the identification of separated peaks was generated using the UPLC− MS/MS system connected to an Xcalibur2 data system (Thermo Scientific Co., USA). Statistical Analysis. Six GL compounds and five root phenotypes of 213 radish roots (3 biological replicates × 71 accessions) were used for principal component analysis (PCA). PCA was performed on the autoscaled GL profiles and five root phenotypes. For the first PCA, levels of six GL compounds were applied and then five root phenotypes combined for the second PCA. Auto-scaling and PCA were conducted using MetaboAnalyst 3.0 (http://www.metaboanalyst. C

DOI: 10.1021/acs.jafc.5b04575 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

IT no.b

na na 102376 102571 250797 na 234952 na 261962 216710 na 32641 na 104268 135584 112254 na na 102371 200374 250758 na 119289 na 250766 200380 234955 250771 na na na na na na 223576 na na 218927 na na 218924 na na

accession

M213 H005 G035 G048 G203 M220 G185 H031 G212 G159 M212 G012 M219 G049 G103 G052 M218 M209 G033 G127 G195 M301 G083 H030 G200 G128 G186 G201 H034 M221 H006 M211 H033 M216 G178 H029 M239-R G173 H001 M239-W G171 M302 H003

KOR KOR KOR ZAF KGZ KOR KOR KOR RUS KOR KOR TUR KOR KOR THA JPN KOR KOR KOR NPL KOR KOR CHN KOR ITA NPL JPN KOR KOR KOR KOR KOR KOR KOR UZB KOR CHN JPN KOR CHN KAZ KOR KOR

origin

11-FD139 13RA-3 JejuWae Nooitgedacht Black Radish E69-1 Pohang-2003 12-FB103 “Ekonom”smes SJeju-2003 WK10041 PI169629 D45-3 Oemusi PI257246 Inyeonja OS-D2382 WK10031 Namwon Am Tokiwase Butdeulmusi Palkwang Nanpanchou 12-FB102 Sango Puthanered RA90 Gegeolmu 12-FB106 D169-2 13RA-4 WK10037 12-FB105 WK10049 UZB-HHS-2008 12-FB100 11-FD162R Sakurajima Giant Gwandong 11-FD162W RA281 Haryeong 13RA-1

name inbred inbred landrace cultivar landrace inbred wild rel. inbred landrace wild rel. inbred cultivar inbred landrace cultivar cultivar inbred inbred landrace wild rel. landrace cultivar wild rel. inbred landrace wild rel. wild rel. landrace inbred inbred inbred inbred inbred inbred landrace inbred inbred landrace cultivar inbred cultivar cultivar inbred

resource ND 246.18 45.02 155.85 451.85 ND 96.48 ND 32.85 209.07 ND 587.25 ND 310.78 153.34 27.35 ND ND 372.83 40.37 89.13 42.06 320.85 ND 55.21 187.19 43.25 25.82 ND 51.98 ND ND ND ND 209.48 ND 96.57 ND ND 94.20 81.69 ND 25.65

GREd

Table 2. Glucosinolate Content (nmol g−1) in Radish Samplesa

51.31 20.27 38.67 222.89

D

25.76 102.06 17.22 13.07

± ± ± ±

± 6.69

± 36.79 ± 64.23

± 30.95

± 73.43

± 33.51

36.35 12.06 27.49 23.79 13.89

± ± ± ± ±

± 105.99 ± 132.79 ± 13.89

± 83.45

± 13.30 ± 84.44

± 60.82

± ± ± ±

± 304.35

182.29 111.94 94.71 52.16 27.07 187.03 69.88 235.17 ND 79.81 286.55 265.18 243.89 148.74 145.96 33.56 164.27 171.48 148.82 58.65 ND 197.16 74.86 103.11 62.97 106.68 85.49 130.34 55.67 194.26 55.68 69.08 ND 23.26 92.77 181.20 21.95 41.16 74.99 88.04 14.35 ND 184.95 34.17 12.93 35.36 30.43 45.70 32.99 76.29 33.37 86.87 26.71 36.79 6.75 27.67 71.46 24.98 15.56 14.26 39.19 4.74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± 19.58

12.73 89.00 96.74 66.31 66.33 96.82 0.00 26.80 97.52 59.31 25.83

± ± ± ± ± ± ± ± ± ± ±

56.85 22.33 32.34 6.15 9.58 66.49 12.13 25.74

± ± ± ± ± ± ± ±

2960.61 1644.70 5294.87 878.52 3216.30 907.26 1258.01 625.94 1826.17 892.27 2086.83 2495.26 1180.56 3172.14 2996.81 769.60 1441.64 1408.96 1303.14 2283.65 2026.86 1394.11 1179.32 464.34 43.03 1190.39 1159.36 588.05 494.59 736.64 456.25 1270.22 2752.73 972.80 423.37 410.88 686.85 1317.08 147.82 738.77 664.92

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 13265.83 10622.16 10768.75 8894.15 8049.87 7879.40 7875.43 7216.30 7189.90 6855.16 6707.37 6044.70 6610.05 6070.57 5962.47 6048.72 5862.95 5737.48 5149.70 5522.57 5419.80 5236.40 4712.09 4926.76 4615.96 4378.49 4440.03 4364.32 4405.13 4207.60 4363.52 4295.48 4135.58 4151.21 3786.69 3808.95 3860.40 3873.85 3803.96 3576.38 3608.65 3673.55 3374.13

GBN

GRH 50.41 35.34 16.98 4.63 32.77 19.00 38.88 30.13 36.82 21.21 ND 19.86 ND 7.75 15.48 4.12 6.57 ND ND ND 7.03 ND ND 20.19 ND ND ND 27.66 9.30 ND 2.88 ND 61.40 ND ND ND ND ND ND ND ND ND ND

21.49 8.14 2.45 0.17 10.72 4.38 16.64 10.70 12.07 0.75

1.22 6.47 0.73 0.84

± 36.45

± 0.69

± 6.01 ± 4.26

± 5.76

± 2.10

± ± ± ±

± 6.38

± ± ± ± ± ± ± ± ± ±

GER ND ND ND ND 22.75 9.08 2.23 46.22 12.17 6.25 ND 18.57 ND 3.09 ND ND ND ND 32.81 ND 16.03 2.22 ND ND ND ND ND 7.24 ND 1.41 7.21 3.59 3.24 18.97 16.95 ND ND ND ND ND ND ND ND 17.00 5.83 0.12 19.41 6.83 0.00

± ± ± ± ± ±

0.21 2.97 2.11 0.00 8.67 8.74

± 1.77

± 10.48 ± 0.26

± 9.85

± 0.11

± 13.46

± ± ± ± ± ±

4HGBS 30.18 12.44 ND 9.25 16.06 15.71 ND 19.90 15.62 ND 23.84 24.30 ND 25.74 ND ND 35.02 47.81 1.97 ND 2.85 ND ND 9.68 ND ND 4.67 2.07 23.87 13.98 2.85 14.63 4.08 9.90 18.09 10.33 ND ND 9.18 ND 2.13 1.78 51.27 1.02 0.17 10.96 7.14 0.51 13.60 0.44 6.15 8.70 2.35

± 0.89 ± 1.04 ± 3.55

± 2.22

± ± ± ± ± ± ± ± ± ±

± 1.81

± 1.37

± 12.64 ± 22.27 ± 0.45

± 10.56

± 7.18 ± 11.20

± 3.99 ± 6.40

± 0.00 ± 4.39 ± 5.30

± 17.93 ± 5.33

4MGBS 13528.72 11028.06 10925.45 9116.03 8600.37 8110.22 8082.9 7547.72 7287.36 7171.5 7017.76 6959.87 6853.93 6566.68 6277.24 6113.74 6068.81 5956.77 5706.12 5621.6 5534.84 5477.84 5107.81 5059.74 4734.14 4672.36 4573.44 4557.45 4493.98 4469.23 4432.15 4382.78 4204.3 4203.34 4123.99 4000.48 3978.92 3915.01 3888.13 3758.61 3706.82 3675.33 3636

total

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

PCAc

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.5b04575 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

E

na na 262086 119288 180726 102484 na 119242 210204 na na 261997 204241 204239 220675 32682 261994 261968 112256 na 250742 218911 234956 na 100715 na 171340 na

H009 H002 G228 G082 G121 G041 M222 G064 G152 M207 M217 G222 G139 G138 G176 G016 G221 G213 G053 H008 G192 G166 G187 M208 G032 M214 G119 H035

KOR KOR PRK AFG EGY IND KOR IRN DEU KOR KOR CHL USA USA UZB IND MYS LBY JPN KOR MMR ITA JPN KOR KOR KOR CHN KOR

origin

13RA-7 Seoho Keijou Arutari PI211745 Egypt wild Mula D232-6 PI138651 Hilds Raxe GS WK10028 B175 Saxa Cherre Belle French Breakfast UZB-HHS-2007 PI181018 Lobak Rondecarlate Hatif Soloechae 13RA-6 MMR-SJS-2011 RA401 RA395 WK10030 Cheongpi Hongsim WK10045 Hongshuiluobo 12-FB107

name inbred cultivar cultivar wild rel. wild rel. cultivar inbred wild rel. cultivar inbred inbred cultivar cultivar cultivar landrace wild rel. cultivar cultivar cultivar inbred landrace wild rel. wild rel. inbred cultivar inbred wild rel. inbred

resource ND ND ND 357.78 125.23 127.04 ND 219.80 49.40 ND 73.56 90.08 140.55 7.28 90.78 8.97 79.55 69.91 275.75 48.44 102.57 181.63 137.60 ND 411.33 36.74 40.42 ND

GREd

19.15 42.87 120.82 1.96 68.44 1.93 30.71 38.89 212.71 22.22 56.43 69.30 29.89

± 133.69 ± 21.85 ± 29.55

± ± ± ± ± ± ± ± ± ± ± ± ±

± 100.21 ± 33.87

± 107.39 ± 103.49 ± 127.53

3450.82 3325.37 3341.52 2826.93 3106.12 2991.66 3245.31 2842.04 2850.50 2879.60 2585.15 2023.01 1809.88 1741.58 1491.61 1360.10 1275.59 973.03 22684.48 22309.01 19060.94 14366.89 9895.94 5344.29 4669.68 4083.73 3835.28 2607.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1869.08 449.04 1253.86 558.32 348.99 2427.69 202.88 2529.35 1566.96 503.96 216.21 317.53 536.55 544.31 511.39 495.18 516.19 466.84 2975.73 4013.80 8557.64 557.32 2581.93 626.88 525.68 910.40 1017.01 619.44

GRH 2.88 125.26 ND 161.94 24.69 ND ND 126.91 140.18 191.53 330.01 ND 60.79 46.08 57.60 ND** ND ND 255.49 209.35 146.45 238.65 ND 405.50 0.00 318.52 50.44 275.49 48.74 0.00 57.16 33.96

187.51 70.96 68.29 84.75 47.13 69.11 69.01 11.97 15.11

± ± ± ± ± ± ± ± ±

± 13.42 ± 6.55 ± 6.59

± ± ± ±

± 74.80 ± 12.17

± 1.42 ± 17.99

GBN ND ND ND ND ND 50.67 ND ND 14.32 ND ND ND ND ND ND ND ND ND 86.87 59.54 153.25 142.43 196.04 0.00 ND ND ND ND ± ± ± ± ± ±

37.01 16.11 91.72 20.50 113.56 10.22

± 6.08

± 13.89

GER ND ND ND ND ND 40.81 ND 5.19 4.02 ND ND 23.67 0.98 ND ND 15.39 8.16 ND ND ND 19.82 9.50 ND 23.95 72.3 72.42 ND 20.67 ± 16.38

± 0.04 ± 29.27 ± 59.24

± 7.88 ± 4.03

± 0.00 ± 2.94

± 0.00 ± 0.47

± 1.78 ± 0.50

± 15.04

4HGBS 16.84 1.39 50.77 ND 8.29 41.51 3.29 1.75 31.40 6.89 2.61 ND 12.44 ND 20.47 ND 14.68 9.00 6.10 42.75 17.90 25.27 3.56 45.46 0.00 69.34 87.59 48.67 2.79 29.41 2.46 0.14 12.42 3.49 1.09

± ± ± ± ± ± ± ± ± ± ± ±

5.59 3.17 1.77 14.29 0.00 10.29 1.15 8.23 14.90 4.51 45.51 3.35

± 10.82

± 5.76

± ± ± ± ± ± ±

± 3.98 ± 0.46 ± 41.62

4MGBS 3470.54 3452.02 3392.28 3346.65 3264.33 3251.7 3248.61 3195.7 3089.81 3078.02 2991.34 2136.76 2024.64 1794.95 1660.46 1384.45 1377.97 1051.94 23308.68 22669.08 19500.93 14964.38 10233.13 5819.19 5153.31 4580.74 4013.72 2951.84

total

I I I I I I I I I I I I I I I I I I II II II II II III III III III III

PCAc

Data are presented as a mean ± SD from three individual roots. bRegistration numbers for http://genebank.rda.go.kr cPCA indicate group numbers from principal component analysis in Figure 3. dND, not detected; na, not available; wild rel., wild relative.

a

IT no.b

accession

Table 2. continued

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.5b04575 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 3. Score (A) and loading (B) plots of PCA for GL profiles in all radish accessions. Three individual samples from the same accession were the same colors for groups II and III. (C) Root phenotypes of ten representative radish accessions from group I (designated as different colored circles for each accession), and five accessions from groups II and III, respectively. ca)23 and XLSTAT (Addinsoft, U.S.A.), respectively. Analysis of variance (ANOVA) and Tukey multiple comparisons were conducted using SPSS v21 (SPSS Inc., U.S.A.). The boxplots were drawn with SigmaPlot 12.0 (Systat Software Inc., U.S.A.).

quantification of GLs because these two GLs were below the detection limit in most of the accessions. Aromatic GLs were not detected in our samples. Radish roots have been reported to have GLs such as GRH, GER, and GRE in common in previous studies.15,17 In addition to these three major GL compounds, we identified four additional GLs: PRO, GBN, 4HGBS, and 4MGBS. GRH was the predominant GL in all the accessions, ranging from 970 to 22 680 nmol g−1 DW and constituting 95.2% of the total GL content on average (Table 2). The total GL content ranged from 1050 to 23 310 nmol g−1 DW, which accounts for 0.04 to 0.98% (w/w) of the total dry weight (Table 2); the highest total GL content among all the accessions was 22 times higher than the lowest total GL content. The highest level of total GLs was observed in accession G053, which is a Japanese cultivar with a conical root shape (Figure 3C). The level of GRH was at least 7.9 times higher than the other GLs in all the accessions examined in this study. The other GLs constituted a very small portion of the total GL content on average, with 2.0% for GRE, 2.2% for GBN, and less than 0.3% for GER, 4HGBS, and 4MGBS. We analyzed GL content in the mature root tissues because the root is the most important edible part of radishes. We could detect only eight GLs in this study out of 15 GLs which have been reported to be present in radish plant.1 Previous studies have shown that the GL profile and content vary by tissue type and developmental stage. For example, the seed showed a

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RESULTS & DISCUSSION Identification of GLs in Radish Roots. We identified eight different GLs, including six aliphatic GLs−glucoerucin (GER), GRH, gluconapin (GNP), glucoraphenin (GRE), progoitrin (PRO), and glucobrassicanapin (GBN), and two indolyl GLs4-hydroxyglucobrassicin (4HGBS) and 4-methoxyglucobrassicin (4MGBS), in the root of radish accession G032 (Table 1 and Figure 2C). For identification of the GLs as the desulfo-GL structure, candidate samples were subjected to LC−ESI−MS analysis in positive and negative modes. The sinigrin standard showed [2MH]−, [MH]−, [MCH2* CHCHNOH], and [S-Glucose] peaks. Most GLs were detected under negative mode at an [MH]− peak, whereas no signal was detected in positive mode as previously reported.21,24 The fragment patterns of desulfo-GLs were dependent on the structure of each GL. GRE, GRH, and GER commonly showed [2M−H]− after desulfatation (Figures S1 and S2). The other GLs, including GBN, PRO, 4HGBS, and 4MGBS, commonly showed [MH]− fragment peaks (Figures S1−S4). The aliphatic GLs in radish roots contain four or five carbons in their side chains. The most prevalent GL in all the samples was clearly GRH, which was biosynthesized from methionine. PRO and GNP were excluded from the F

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Figure 4. PCA for GL profiles and root phenotypes. (A) Score plot showing that the radish accessions are putatively categorized into four groups named groups i, ii, iii, and iv based on their GL profiles and root phenotypes. Three individual samples from the same accession were same colored for groups ii, iii, and iv. (B) Loading plot of six GL contents and five root phenotypes. (C) Root phenotypes of four radish accessions from group iii, and six from group iv.

different GL profile from that of the vegetative tissue, displaying the highest level of GLs in Arabidopsis.20 A total of 15 GLs have been identified in the whole radish plant, including the eight GLs identified from radish roots in this study.1 Many of the minor GLs were identified from seeds or seedlings, and it is possible that the major GLs are primarily stored in the storage tissue: the roots in radish plants. R. sativus has Distinct GL Profiles from B. rapa. Two accessions of turnip (B. rapa), one from Russia and the other from Korea, were analyzed; they were grown, sampled, and analyzed simultaneously with the radish accessions. Most of the GLs detected in the turnip samples were the same as those found in a previous report.9 The total amount of GL in these two turnips was below the average GL amount found in the 71 radish accessions. Two clear differences were observed between the radish and turnip samples. First, GRE and GRH were R. sativus-specific GLs, whereas gluconasturtiin was only detected in B. rapa; many GLs, such as PRO, GNP, GBN, GER, and 4MGBS, coexisted in both turnips and radishes (Table S1).

Second, aliphatic GLs were predominant in the total GL composition of R. sativus, which was at least 97%; a larger amount of indolyl GL was observed in the two B. rapa accessions, G157 and G183 with 55% and 29% of the total GL content, respectively (Table S2). These differences confirmed that there are genotype-specific GL profiles in R. sativus and B. rapa. These genotype-specific profiles could be used to distinguish radishes from turnips, which is often difficult based on the phenotype alone. Overall, the GL profiles of 71 radish accessions suggest that GL biosynthesis and accumulation in radish roots had a common pathway despite the accessions originating from different countries; these pathways are clearly distinguishable from other known Brassica species. However, the predominance of aliphatic GLs in radish roots was similar to other Brassica family species. Aliphatic GLs are known to be transported mainly to major storage organs such as rosettes and roots after biosynthesis in Arabidopsis.25 The site of biosynthesis of the GLs stored in radish roots is still unknown. G

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Journal of Agricultural and Food Chemistry Principal Component Analysis for GL Profiles Reveals Three Different Groups. PCA was employed to evaluate and differentiate the radish accessions on the basis of GL content and profiles. As shown in Figure 3A, PC1 and PC2 account for 55.70% of all the variables. The six GL contents were positively loaded to PC1 with the highest loadings of GRH and GER. PC2 is composed of three positive loadings, 4MGBS, 4HGBS, and GBN, and three negative loadings, GRH, GER, and GRE (Figure 3B). The radishes could be separated into three groups when projected to a two-dimensional plot of PC1 and PC2 (Figure 3A), indicating variations in the GL profiles of these radish samples. Group I, which contained 61 radish accessions out of 71 accessions, was located in the middle of the plot, within ±2 for PC1 and ±1.7 for PC2. Group II was bigger than 2 for PC1 with negative for PC2 or positive for PC1 with below −2 for PC2. Group II includes five radish accessions, which have higher amounts of GRH and GER. Because GRH is the most prominent GL, the radishes in group II ranked highest in terms of the total amount of GL. Group III was positive for PC1 and bigger than 1.8 for PC2. Group III was characterized by a relatively high amount of 4HGBS or 4MGBS. Four of them also contained a high amount of GBN at the same time. Two accessions (G187 and G166) in group II showed similar root phenotypes, with a small triangular shape and many secondary roots (Figure 3C). The radish accessions in group I varied in their shapes, colors, weight, and pithiness. Seven accessions showed inconsistent locations for three biological replicates, suggesting their genetic heterogeneity or environmental effects on GL profile. The highest correlation was observed between the amount of GRH and GER using Pearson’s correlation coefficient 0.682 (p < 0.001). The correlation between two aliphatic GLs, GRH, and GER, was also reported in nine Brassicaceae crops, including radish.26 These two GLs are the first and the fourth highest GL on average in the 71 accessions (Table 2). Both GLs are aliphatic and share methionine as a precursor. However, not all the aliphatic GLs showed a correlation, suggesting an underlying complex regulatory mechanism in the GL biosynthesis pathway. The other significant correlation was observed between 4MGBS and GBN (0.323, p < 0.01) and between 4MGBS and 4HGBS (0.317, p < 0.01). 4MGBS and 4HGBS are both indolyl GLs from the same precursor. The correlation between 4MGBS and GBN suggests possible coregulation of aliphatic and indolyl biosynthesis pathways. To further investigate the relationship between GL profile and root phenotype, five root phenotypes including shape, tissue pithiness, sweetness, exterior color, and length were analyzed. None of these root phenotypes showed a strong correlation with GL content. An additional PCA with these five root phenotypes and GL profiles generated four groups (Figure 4A). PC1 and PC2 accounted for 38.47% of all the variables. The largest group (group i) was located in the middle of the plot, containing 57 accessions between +1.9 and −2.6 for PC1 and between +1.7 and −2.3 for PC2. Members of group ii include four from group II and one from group III in the first PCA, with a high GL content, especially for GRH, GER, and GBN. The radish accessions in group iii showed a high amount of GRE and dark exterior color. Group iv contained six accessions, which have a low amount of GL and are characterized by a short and spherical shape. Members of group iv have a pink exterior color and weak pithiness. These PCA results showed there were at least three different genotypes related to GL profiles in radish accessions, which

were categorized into groups I to III. Additional molecular genetic studies are required to determine the differences between these genotypes and their origins. It is notable that three dark-colored radish accessions, either brown or black, had a high GL content and were grouped together. The other notable observation in exterior color phenotype was that pinkor red-colored radish accessions showed a lower GL content. There were 11 radish accessions, including six accessions in group iv, that had a pink or red exterior color. The average the total GL amount of these 11 accessions was only 64.5% of the average of all the accessions. In general, root phenotypes such as shape, length, and pithiness do not have a strong relationship with the GL profiles of radish accessions, suggesting that GL content and variation could not be expected based only on root phenotype. Furthermore, GL quantification is required to select high GL accessions. Simple analysis methods and molecular makers based on genetic analysis are necessary for high GL breeding programs. However, because there is no strong correlation between phenotype and GL profile, high GL radish cultivars of various shapes and sizes could be bred. Difference in the Variation of Landraces, Cultivars, and Wild Relatives. The 71 radish accessions could be categorized by geographical origin and degree of domestication. The accessions were collected from 23 different countries and grouped based on their continental origin. Accessions obtained from northeast Asia (n = 47), southern or central Asia (n = 16) and other areas (n = 8) including Europe, Africa, and America were compared using ANOVA. No significant difference was found among these three groups, with the exception of the high amount of GRE in accessions from central and southern Asia (Figure S5). High GRE accessions were also separately grouped as group iii in the PCA (Figure 4A). Two accessions (G012 and G203) of group iii originated from central Asia. Because GRE is comprised of a small portion of total GL content, there was no geographical difference in the total GL amount. The samples included 13 wild relatives, 12 landraces, 19 cultivars, and 27 inbred lines. On the basis of the ANOVA results for the four categories, significant differences were found in GRE content and GBN content for the inbred line population, in which GRE was significantly low and GBN was significantly high. However, major GL, GRH, and total GL content were similar in all groups. Regarding the total GL content, there were no differences among wild relatives, landraces, and cultivars (Figure S6). In addition, the GL content was not significantly different in 5 landraces from different regions in Korea and 5 commercial cultivars in Korea. This suggests that the level of GL has not been considered as a breeding target. There were eight accessions with over 10 000 nmol g−1 DW of total GL, including two wild relatives, two landraces, one cultivar, and three inbreds. These results suggest that breeding high GL-containing radish cultivars is still exploitable, and high levels of the GL germplasm could come from any type of genetic resource. In this study, we analyzed GL content and profiles in radish roots for 71 accessions and showed the specificity and variation of GL profiles in radish roots. To date, only a few GLs have been confirmed for their health-promoting activity. High-GL containing radish would be accomplished by increasing the major GL, GRH. However, if different GLs have different activity or combined GLs have a synergistic effect for human health, more complex breeding strategies would be required. For any cases, an understanding of the genetic variation in GL H

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(5) Nakamura, Y.; Nakamura, K.; Asai, Y.; Wada, T.; Tanaka, K.; Matsuo, T.; Okamoto, S.; Meijer, J.; Kitamura, Y.; Nishikawa, A.; Park, E. Y.; Sato, K.; Ohtsuki, K. Comparison of the glucosinolatemyrosinase systems among daikon (Raphanus sativus, Japanese white radish) varieties. J. Agric. Food Chem. 2008, 56, 2702−2707. (6) Okamura, T.; Umemura, T.; Inoue, T.; Tasaki, M.; Ishii, Y.; Nakamura, Y.; Park, E. Y.; Sato, K.; Matsuo, T.; Okamoto, S.; Nishikawa, A.; Ogawa, K. Chemopreventive effects of 4-methylthio-3butenyl Isothiocyanate (Raphasatin) but not curcumin against pancreatic carcinogenesis in hamsters. J. Agric. Food Chem. 2013, 61, 2103−2108. (7) Papi, A.; Farabegoli, F.; Iori, R.; Orlandi, M.; De Nicola, G. R.; Bagatta, M.; Angelino, D.; Gennari, L.; Ninfali, P. Vitexin-2-O-xyloside, raphasatin and (−)-epigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells. Food Chem. 2013, 138, 1521−1530. (8) Farnham, M. W.; Stephenson, K. K.; Fahey, J. W. Glucoraphanin level in broccoli seed is largely determined by genotype. Hortscience 2005, 40, 50−53. (9) Lee, J. G.; Bonnema, G.; Zhang, N.; Kwak, J. H.; de Vos, R. C.; Beekwilder, J. Evaluation of glucosinolate variation in a collection of turnip (Brassica rapa) germplasm by the analysis of intact and desulfo glucosinolates. J. Agric. Food Chem. 2013, 61, 3984−3993. (10) Hasan, M.; Friedt, W.; Pons-Kuhnemann, J.; Freitag, N. M.; Link, K.; Snowdon, R. J. Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus). Theor. Appl. Genet. 2008, 116, 1035−1049. (11) Ramos, S. J.; Yuan, Y.; Faquin, V.; Guilherme, L. R.; Li, L. Evaluation of genotypic variation of broccoli (Brassica oleracea var. italic) in response to selenium treatment. J. Agric. Food Chem. 2011, 59, 3657−3665. (12) Wiesner, M.; Zrenner, R.; Krumbein, A.; Glatt, H.; Schreiner, M. Genotypic variation of the glucosinolate profile in pak choi (Brassica rapa ssp. chinensis). J. Agric. Food Chem. 2013, 61, 1943−1953. (13) Ishida, M.; Hara, M.; Fukino, N.; Kakizaki, T.; Morimitsu, Y. Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables. Breed. Sci. 2014, 64, 48−59. (14) Ediage, E. N.; Di Mavungu, J. D.; Scippo, M. L.; Schneider, Y. J.; Larondelle, Y.; Callebaut, A.; Robbens, J.; Van Peteghem, C.; De Saeger, S. Screening, identification and quantification of glucosinolates in black radish (Raphanus sativus L. niger) based dietary supplements using liquid chromatography coupled with a photodiode array and liquid chromatography-mass spectrometry. J. Chromatogr., A 2011, 1218, 4395−4405. (15) Ishida, M.; Nagata, M.; Ohara, T.; Kakizaki, T.; Hatakeyama, K.; Nishio, T. Small variation of glucosinolate composition in Japanese cultivars of radish (Raphanus sativus L.) requires simple quantitative analysis for breeding of glucosinolate component. Breed. Sci. 2012, 62, 63−70. (16) Malik, M. S.; Riley, M. B.; Norsworthy, J. K.; Bridges, W., Jr. Glucosinolate profile variation of growth stages of wild radish (Raphanus raphanistrum). J. Agric. Food Chem. 2010, 58, 3309−3315. (17) Ishida, M.; Kakizaki, T.; Ohara, T.; Morimitsu, Y. Development of a simple and rapid extraction method of glucosinolates from radish roots. Breed. Sci. 2011, 61, 208−211. (18) Kiddle, G.; Bennett, R. N.; Botting, N. P.; Davidson, N. E.; Robertson, A. A. B.; Wallsgrove, R. M. High-performance liquid chromatographic separation of natural and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and chemical ionisation-MS methods. Phytochem. Anal. 2001, 12, 226−242. (19) Bennett, R. N.; Mellon, F. A.; Kroon, P. A. Screening crucifer seeds as sources of specific intact glucosinolates using ion-pair highperformance liquid chromatography negative ion electrospray mass spectrometry. J. Agric. Food Chem. 2004, 52, 428−438. (20) Brown, P. D.; Tokuhisa, J. G.; Reichelt, M.; Gershenzon, J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 2003, 62, 471−481.

profiles would be helpful for breeding specific GL-containing radishes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04575. Table S1, Glucosinolates detected in turnip roots; Table S2, glucosinolate content (nmol g−1 DW) in turnip samples; Figure S1, mass chromatograms of aliphatic GLs; Figure S2, mass spectrum patterns of aliphatic GLs; Figure S3, mass chromatograms of indolyl GLs; Figure S4, mass spectrum patterns of indolyl GLs; Figure S5, box plots of each GL content based on their geographical origin of radish accessions; and Figure S6, box plots of each GL content based on their degree of breeding (DOCX)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +82-2-880-4562; fax: +82-2-873-2056; e-mail: gibumyi@ gmail.com (G.Y.). *Tel: +82-2-880-4562; fax: +82-2-873-2056; e-mail: huhjh@ snu.ac.kr (J.H.H.). Author Contributions ⊥

These authors contributed equally to this work.

Funding

This study was supported by a grant from the Golden Seed Project (213002041SBO20), Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea. Sooyeon Lim was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2013R1A1A1057658 to E.J.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Suhyoung Park for providing the radish inbred line samples. ABBREVIATIONS GL, glucosinolate; ITC, isothiocyanate; ANOVA, analysis of variance; PCA, principal component analysis; GER, glucoerucin; GRH, glucoraphasatin; GNP, gluconapin; GRE, glucoraphenin; PRO, progoitrin; GBN, glucobrassicanapin; 4HGBS, 4-hydroxyglucobrassicin; 4MGBS, 4-methoxyglucobrassicin

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REFERENCES

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