Genotypic variation of glucosinolates and their breakdown products in

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Agricultural and Environmental Chemistry

Genotypic variation of glucosinolates and their breakdown products in leaves of Brassica rapa Rebecca Klopsch, Katja Witzel, Anna Artemyeva, Silke Ruppel, and F Hanschen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01038 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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Journal of Agricultural and Food Chemistry

Genotypic Variation of Glucosinolates and their Breakdown Products in Leaves of Brassica Rapa

Rebecca Klopsch1, Katja Witzel1, Anna Artemyeva2,3, Silke Ruppel1, and Franziska S. Hanschen1*

1

Leibniz Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1,

14979 Großbeeren, Germany 2

N.I.Vavilov Institute of Plant Genetic Resources, Bolshaya Morskaya Street 42-44,

190000 St. Petersburg, Russia 3

Agrophysical Research Institute, Grazhdanskiy prospect 14, 195220 St. Petersburg,

Russia *

Corresponding author:

Franziska S. Hanschen Leibniz Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany Tel: 0049-33701-78241 Fax: 0049-33701-55391 Email: [email protected]

ACS Paragon Plus Environment


1

Abstract

2

An in-depth glucosinolate (GLS) profiling was performed on a core collection of 91

3

Brassica rapa accessions, representing diverse morphotypes of heterogenous

4

geographical origin, to better understand the natural variation in GLS accumulation

5

and GLS breakdown product formation. Leaves of the 91 B. rapa accessions were

6

analyzed for their GLS composition by UHPLC-DAD and the corresponding

7

breakdown products by GC-MS. Fifteen different GLSs were identified and aliphatic

8

GLSs prevailed regarding diversity and concentration. Twenty-three GLS breakdown

9

products were identified, among them nine isothiocyanates, ten nitriles and four

10

epithionitriles. Epithionitriles were the prevailing breakdown products due to the high

11

abundance of alkenyl GLSs. The large scale data set allowed the identification of

12

correlations in abundance of specific GLSs or of GLS breakdown products.

13

Discriminant function analysis identified subspecies with high levels of similarity in the

14

acquired metabolite profiles. In general, the five main subspecies grouped

15

significantly in terms of their GLS profiles.

16 17

Key Words: Isothiocyanates, epithionitriles, nitriles, plant secondary metabolites,

18

genotypic variation

19 20 21 22 23 24 25 26 ACS Paragon Plus Environment

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27

Introduction

28

Natural variation in crops represents an important tool for plant improvement. Crop

29

breeding relies on plant genetic diversity. Recently, genome sequencing projects,

30

linkage

31

understanding of complex traits.1-3 While natural variation is often studied in respect

32

to phenotypic variation, until now little attention has been paid to the diversity in

33

chemical composition of plants and how this can be integrated into genetic

34

analyses.4,

35

prerequisite for such genetic analyses.6

36

Brassica vegetables are cultivated worldwide and high consumption of Brassica

37

vegetables (e.g. B. rapa, B. oleracea, B. napus) correlates negatively to

38

carcinogenesis in epidemiological studies.7,

39

credited to glucosinolates (GLSs), a group of secondary plant metabolites almost

40

exclusively found in plants of the order Brassicales. Over the last decades mainly the

41

aliphatic GLSs and their breakdown products were in the scientific focus.9, 10

42

For example, sulforaphane (4-(methylsulfinyl)butyl ITC) has been studied extensively

43

in vitro and in vivo including its effect on enzymes of biotransformation and

44

cytoprotection, responsible for the anticarcinogenic effects.11-13

45

GLSs can be classified according to their respective amino acid precursors such as

46

Ala, Val/Leu, Ile, Met, Phe/Tyr, and Trp.14 Aliphatic GLSs principally derive from Met,

47

while among the aromatic GLS, the indole GLSs derive from Trp and arylaliphatic

48

GLSs mostly derive from Phe.14 More than 50% of the GLSs identified so far are

49

aliphatic with highly variable side chain structures.14 The biosynthesis proceeds

50

through three separate biosynthetic steps: 1) the chain elongation of selected

51

precursor amino acids (relevant for Met- and Phe-derived GLSs); 2) the formation of

mapping

5

and

genome-wide-association

studies

led

to

a

deeper

Elaborate metabolite screens of a large number of genotypes are a

8

The health-promoting effects were

1



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52

the GLS core structure, and 3) modifications of the side chain.15 In plants,

53

synthesized GLSs can accumulate in specialized S-cells16 while the GLS-hydrolyzing

54

myrosinases (β-D-thioglucosidases) can be located in different types of “myrosin

55

cells”16, 17 and can be located in different cell compartments such as the vacuole or

56

endoplasmic reticulum bodies.18, 19 Upon cell rupture, myrosinase comes into contact

57

with the GLS and enzymatic degradation is initiated (Figure 1), forming a variety of

58

breakdown products. In a first step, the enzyme cleaves the GLS and releases β-D-

59

glucose and an unstable thiohydroximate-O-sulfate. Then, the aglucon transforms to

60

the ITC via a Lossen-like rearrangement, or decomposes to the corresponding nitrile.

61

Some plants modulate this transformation by specific proteins, such as the

62

epithiospecifier protein (ESP), the nitrile specifier protein (NSP) or the thiocyanate-

63

forming protein (TFP).20 The ESP protein promotes the formation of epithionitriles

64

(EPT) from unsaturated aliphatic GLSs and of nitriles from non-alkenyl GLSs. With

65

regard to the hydrolysis products formed, especially ITCs are responsible for their

66

antimicrobial, antifungal, and antiherbivorous properties in several Brassica

67

vegetables.21, 22

68

Brassica species have enormous genetic and morphological diversity, which

69

contributes to the great diversity in adaptation to biotic and abiotic stresses. Variance

70

in GLS concentrations has been extensively studied in B. rapa, either in turnips,23, 24

71

leaves,25-32 or both,33, 34 underlining a high variance in absolute GLS content as well

72

as in its composition. Application of GLS metabolite profiling to map underlying

73

genetic factors in a doubled haploid B. rapa population recently allowed the allocation

74

of metabolic and transcript expression QTL of the GLS pathway.35 However,

75

observed bioactive effects due to GLSs originate mainly from their breakdown

76

products. Only recently the scientific focus has shifted towards profiling GLS 2


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77

breakdown products of B. rapa accessions in order to precisely select accessions

78

suitable to promote human health or for sustainable plant production by herbivore

79

resistant accessions.33, 36

80

Here, we evaluated the profile and quantity of both GLSs and their respective

81

breakdown products in a core collection of 91 B. rapa accessions from the Russian

82

VIR gene bank. These accessions have been previously used for an association

83

mapping approach,37 and are well described for morphological and biochemical

84

traits.38 Table 1 provides an overview over the compounds investigated. The current

85

study aims to enlarge the genetic diversity of Brassica species used in food

86

production by generating detailed information about the plant metabolite composition

87

and concentration in order to enable a more targeted crossing and breeding.

88

Furthermore, we assessed the correlation between the abundance of the various

89

GLSs and their breakdown products to gain a deeper understanding of the interactive

90

effects resulting in differential metabolite accumulation.

3



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91

Materials and Methods

92

Chemicals

93

Methanol (≥ 99.9%) and acetonitrile (≥ 99.9%) were from Fisher Scientific GmbH,

94

Schwerte, Germany; acetic acid (≥ 99.9%) and arylsulfatase were from Merck KGaA,

95

Darmstadt, Germany; formic acid (≥ 98%), imidazole (≥ 99%), 4-hydroxybenzyl GLS

96

(≥ 99%), methylene chloride (GC Ultra Grade), and 2-propenyl GLS (2,

97

ROTICHROM® CHR) were from Carl Roth GmbH, Karlsruhe, Germany; benzonitrile

98

(≥ 99.9%, 3-(methylthio)propyl ITC (≥98%), 4-pentenenitrile (3b, ≥97%), 3-

99

phenylpropanenitrile (11b; ≥99%), and 2-phenylethyl isothiocyanate (11a, ≥99%)

100

were purchased from Sigma-Aldrich Chemie GmbH, Darmstadt, Germany;

101

anhydrous Na2SO4 (≥ 99%) was obtained from VWR International GmbH, Darmstadt,

102

Germany; 3-butenyl GLS (3), 2-(R)-2-hydroxy-3-butenyl GLS (4; ≥98%), 4-

103

(methylsulfanyl)butyl GLS (7, ≥98%), 3-(methylsulfinyl)propyl GLS (9, ≥98%), 4-

104

(methylsulfinyl)butyl GLS (10, ≥98%), and 2-phenylethyl GLS (11, ≥98%) were

105

obtained from Phytolab GmbH & Co. KG, Vestenbergsgreuth, Germany; 4,5-

106

epithiopentanenitrile (3c, ≥ 99%) and 5,6-epithiohexanenitrile (4c, ≥ 99%) were

107

purchased from ASCA GmbH Angewandte Synthesechemie Adlershof, Berlin,

108

Germany; 3-indoleacetonitrile (12b) (≥98%) was acquired from Fischer Scientific

109

GmbH, Schwerte, Germany; 3-Butenyl ITC (3a, ≥95%) and 1-methylpropyl ITC (1a,

110

≥98%) were obtained from TCI Deutschland GmbH, Eschborn, Germany; 4-

111

(methylthio)butyl ITC (7a, ≥98%), was purchased from Santa Cruz Biotechnology,

112

Heidelberg, Germany. 4-(methylsulfinyl)butyl ITC (10a, ≥98%) was bought from Enzo

113

Life Sciences GmbH, Lörrach, Germany; 5-vinyloxazolidine-2-thione (4a) was

114

purchased from Biosynth AG, Staad, Switzerland; 1-Methoxyindole-3-carbinol (13a)

115

and 1-methoxyindole-3-acetonitrile (13b) were a kindly gift from H.R. Glatt (German 4


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116

Institute of Human Nutrition, Department of Nutritional Toxicology, Potsdam-

117

Rehbrücke, Germany); 5-Hexenenitrile (5b) was a gift of S. Rohn (University of

118

Hamburg). All solvents were of LC-MS or GC-MS grade, water was of Milli-Q quality.

119 120

Plant material

121

Seed material of 91 B. rapa accessions originating from the Russian ex-situ

122

germplasm collection located at the N.I. Vavilov All Russian Institute of Plant Genetic

123

Resources (VIR). The majority of these accessions originated from the Eurasian

124

area.

125

Seeds were sown in single compartments (3x3 cm) of breeding shells filled with soil

126

(Einheitserde

127

Germany) and then grown in a climate chamber under controlled conditions.

128

Temperature settings were 22°C during the day and 20°C at night, air humidity was

129

65%, light intensity was 400 µmol m-2 s-1 (metal halid lamp, EYE Clean Ace

130

(MT400DL/BH) EYE Lighting Europe Ltd ©, Middlesex, United Kingdom), and the

131

photoperiod was 12/12 h. Plants were watered once a day on demand with tap water,

132

no fertilizer was added. After 3 weeks, at uniform harvest times between 09:00 and

133

10:00 a.m., the leaves of the plants were harvested (3-6 leaf stage). Six plants per

134

accession were harvested for their leaves (except oldest and youngest leaf). In each

135

accession, three replicas comprising two plants were formed. GLS breakdown

136

product extraction was carried out using fresh plant material (300 mg per replica)

137

immediately after harvesting. For GLS analysis the remaining plant material was

138

frozen instantly in liquid nitrogen and lyophilized later on.

classic,

Einheitserde

Werkverband

e.V.,

Sinntal-Altengronau,

139 140

Extraction and analysis of GLS as desulfo-GLS from leaves 5



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141

To detect desulfo-GLS, the methods of Wiesner et al.30 and Witzel et al.39 were

142

adapted (both based on DIN EN ISO 9167-1).40 Briefly, 10 mg of lyophilized and

143

ground plant tissue were extracted using 70°C hot 70% methanol. For quantitation

144

0.5 µmol 4-hydroxybenzyl GLS was added as an internal standard. After 3

145

extractions the combined extracts were loaded onto DEAE-Sephadex A-25 ion-

146

exchanger columns. Extracts were desulfated using aryl sulfatase afterwards the

147

desulfo-GLS were eluted with deionized water (1 mL). Analytes were analyzed using

148

a UHPLC Agilent 1290 Infinity System (Agilent Technologies, Waldbronn, Germany)

149

equipped with a Poroshell 120 EC-C18 column (dimension: 100 mm x 2.1 mm, 2.7

150

µm; Agilent Technologies). Analytes were separated using a gradient of water and

151

acetonitrile, and quantitated at 229 nm with the internal standard. Individual desulfo-

152

GLS were identified by comparison of their retention times and mass spectra with

153

individual desulfo-GLS in standard reference materials of oilseed rape (BCR-190R

154

and BCR-367 R) and with analytical standards.41, 42 The response factors reported in

155

the DIN EN ISO 9167-1 were used for quantitation and were recalculated for 4-

156

hydroxybenzyl GLS after determination of its response factor relative to GLS 2.40

157 158

Extraction and analysis of GLS breakdown products from leaves

159

The enzymatically formed degradation products of GLS were detected using the

160

method of Klopsch et al.33 Deionized water was added to the fresh leaf material

161

(plant:water 1:1). These samples were homogenized in a vibratory mill (MM400

162

Retsch GmbH, Haan, Germany) for 2 min with 4 metal balls (Ø 5 mm) at 30 Hz. An

163

aliquot of the plant material was transferred into a solvent resistant vessel and GLS

164

hydrolysis products were extracted and analyzed as reported by Hanschen et al.

165

(2017).43 For GLS breakdown product extraction methylene chloride was used. For 6


Page 9 of 34


166

quantitation 100 µL of the internal standard benzonitrile solution (2 mM, equates 0.2

167

µmol) were added. After two extractions the samples were dried with anhydrous

168

sodium sulfate, concentrated under nitrogen gas flow to 300 µL, transferred into a

169

vial and analyzed by GC-MS.33 Analyte content was calculated as described

170

previously by using benzonitrile as internal standard and the response factors

171

calculated from the ratio of the slope of linear calibration curves (R2 ≥0.97) relative to

172

that of the internal standard.33 For the commercially unavailable compounds, a

173

response factor equal to that of the chemically most similar compound was assumed:

174

the nitriles 3-methylpentanenitrile (1b), 5-(methylsulfinyl)pentanenitrile (7b), and 5-

175

(methylsulfinyl)pentanenitrile

176

corresponding ITC, 3-hydroxypentenenitrile (4b) was calculated using the RF of 3b,

177

6-(methylsulfinyl)hexanenitrile (8b) was calculated using the RF of 7a and the EPTs

178

3-hydroxy-4,5-epithiopentanenitrile (4c) and 3-hydroxy-5,6-epithiohexanenitrile (6c)

179

were calculated using the RF of 3c.

(10b)

were

calculated

with

the

RF

of

their

180 181

Data analysis

182

Discriminant function analysis (DFA) of GLS and GLS breakdown product profiles of

183

the five main B. rapa subspecies (B. rapa ssp. chinensis, B. rapa ssp. dichotoma, B.

184

rapa ssp. oleifera f. annua, B. rapa ssp. pekinensis, B. rapa ssp. rapa) was

185

performed. Therefore, the measured concentrations of all GLS and GLS breakdown

186

products in 66 B. rapa accessions were included in the analysis. DFA of GLS

187

breakdown products 4MSOB-ITC, 4MSOB-CN, 5MTP-ITC, 1MOI3C, 1MOIAN was

188

not possible due to a limited detection in some accessions. DFA was conducted by

189

Statistica 64 (StatSoft (Europe) GmbH, Germany) in stepwise forward reduced mode.

7



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190

A Pearson correlation was conducted using SigmaPlot 13.0 (Systat Software GmbH,

191

Erkrath, Germany). For correlation analysis, depending on the presence of the

192

respective compound, observations ranging from 91 to 4 for GLS profiles (see

193

Supplemental Table S2) and 89 to 1 for GLS breakdown product profiles (see

194

Supplemental Table S3).

195

196

Results and Discussion

197

Variation in GLS profiles among B. rapa accessions

198

Profiling of GLS metabolites was conducted in leaves of 91 B. rapa accessions to

199

complement a recent evaluation of this collection on morphological, biochemical, and

200

genetic factors.38 The leaves of these accessions showed great variety in size,

201

shape, color, and texture. The average fresh weight (FW) differed from 1.00 g in VIR

202

P-143 to 8.48 g in VIR 132. Quantitative analysis of the GLS profile identified 15

203

different GLSs. This diversity underlines the variability in the GLS biosynthesis

204

pathway, enabling constant adaptation to the surrounding conditions.10, 15 Of the ten

205

aliphatic

206

glucocochlearin, only tentatively identified). Nine derived from Met: 2-propenyl (2,

207

sinigrin), 3-butenyl (3, gluconapin), 2-(R)-2-hydroxy-3-butenyl (4, progoitrin), 4-

208

pentenyl (5, glucobrassicanapin), 2-hydroxy-4-pentenyl (6, gluconapoleiferin), 4-

209

(methylsulfanyl)butyl (7, glucoerucin), 5-(methylsulfanyl)pentyl (8, glucoberteroin), 3-

210

(methylsulfinyl)propyl (9, glucoiberin), and 4-(methylsulfinyl)butyl (10, glucoraphanin).

211

The remaining five GLSs were aromatic, thereof one was Phe derived: 2-phenylethyl

212

(11, gluconasturtiin), and four were Trp derived: 3-indolylmethyl (12, glucobrassicin),

213

1-methoxy-3-indolylmethyl (13, neoglucobrassicin), 4-methoxy-3-indolylmethyl (14, 4-

GLSs

one

was

derived

from

Leu,

namely

1-methylpropyl

(1,

8


Page 11 of 34


214

methoxyglucobrassicin),

215

hydroxyglucobrassicin). The detected GLSs and their detected corresponding

216

breakdown products provided in Table 1. The total GLS concentration ranged from

217

0.02 µmol g-1 FW (VIR 111) to 4.97 µmol g-1 FW (VIR 242) (Figure 2). The plants

218

investigated were six weeks old and had developed three to six leaves. Accordingly

219

there were some variations in the maturity between different accessions. Differing

220

GLS concentrations, and thereby differing GLS breakdown product concentrations,

221

could be driven by these differences as leaves that initiated earlier in plant

222

development could have lower GLS concentrations compared to leaves that initiated

223

later in plant development.44 Overall, GLSs 3 (0 to 3.67 µmol g-1 FW) and 5 (0.003 to

224

1.63 µmol g-1 FW) were the main GLSs. GLS 3 was detected in 87 and 5 in all

225

accessions investigated. So were the indole GLSs (12, 13, 14, 15), however their

226

concentrations in the plant tissue were rather low. The main GLS 3 was found in

227

highest amounts in accessions VIR 96, VIR P-143, VIR 115 and VIR 242 (1.52 to

228

3.67 µmol g-1 FW). Leaves of VIR 242, VIR 13, VIR 161 and VIR 338 accumulated

229

the highest content of GLS 5 (0.87 to 1.63 µmol g-1 FW). A similar comprehensive

230

study analyzed 113 different varieties of turnip greens (B. rapa) for their GLS profile

231

and identified 3 and 5 as the predominant GLSs. In accordance with our results, GLS

232

11 was the only arylaliphatic GLS identified.29 Comparative results were obtained in

233

other studies on B. rapa subspecies.24,

234

revealed high variations in the aliphatic GLSs 3 and 5, and the indole GLS 12 to be

235

dominant in B. rapa.28-30, 33

236

Artemyeva et al.

237

chlorophyll, ascorbic acid and carotenoid content, depending on growing conditions

238

in the studied B. rapa collection. To check whether a correlation exists between fresh

38

and

4-hydroxy-3-indolylmethyl

28-30, 33, 45

(15,

4-

In general, those screenings

previously found correlations between shoot dry matter and

9



Page 12 of 34

239

weight and GLS concentration, a Pearson correlation analysis was performed. A

240

minor positive correlation between fresh weight and GLS concentration was found (r

241

= 0.221, p = 0.036). The extensive data set of GLS profiles in the present study

242

enabled the performance of Pearson correlation analysis testing the co-abundance of

243

GLSs showing that significant correlations were always positive (Table 2). Highly

244

significant correlations (p ≤ 0.001) in the abundance of GLSs were detected among

245

Met-derived aliphatic GLSs (3 and 5, 4 and 6, 3 and 10) probably because their

246

biosynthesis is driven by the same amino acid precursor.9 The strong dependence

247

between dihomo-methionine- and trihomo-methionine-derived GLSs (3 and 5, 4 and

248

6) indicates that GLS side chain elongation is genetically conserved.46,

249

oxoglutarate-dependent dioxygenase coding BrAOP genes are involved in the side

250

chain modifications of aliphatic GLSs.48 Strong significant correlations were found for

251

4 and 6 and point to a conserved conversion from methylsulfinylalkyl GLSs to the

252

corresponding alkenyl GLSs. Further, we found a high positive correlation between

253

two indole GLSs (12 and 15) and the aliphatic 6. Pfalz et al.

254

AOP locus is involved in accumulation of aliphatic as well as indole GLSs. In

255

addition, MYC2 was shown to be involved in both aliphatic and indole GLS

256

formation.50 The high correlation between indole GLSs 12 and 15 (and 12 and 14)

257

could be explained by linked biosynthesis pathways: 12 as the precursor for all other

258

indole GLS is formed by the amino acid Trp in the core biosynthetic pathway and can

259

further be transformed into other indole GLSs by hydroxylation and methylation.9

260

Then, as shown for Arabidopsis, 15 is formed by hydroxylation of 12 catalyzed by

261

CYP81F1, CYP81F2, and CYP81F3, which can be further modified to 14 by O-

262

methyltransferases.51

49

47

The 2-

suggested that the

10


Page 13 of 34


263

264

GLS breakdown product profile and degradation pattern of ITCs, EPTs and nitriles

265

The GLS degradation product profiles and concentrations revealed a high diversity

266

among the 91 B. rapa accessions, similar to an earlier screening of 16 B. rapa turnip

267

accessions.33 Corresponding degradation products of all GLSs found were identified

268

in the homogenized tissues, with two exceptions (breakdowns from 2 and 9).

269

Altogether 23 different GLS breakdown products were identified (Compounds marked

270

with

271

by comparing their EI mass spectra with the literature.). Nine of them could be

272

assigned to the group of ITCs or products thereof: sec-butyl ITC (1a), 3-butenyl ITC

273

(3a),

274

(methylsulfanyl)butyl

275

(methylsulfinyl)butyl ITC (10a), 2-phenylethyl ITC (11a) and 1-methoxyindole-3-

276

carbinol (13a); ten to the group of nitriles: 2-methylbutanenitrile (1bx), 4-pentenenitrile

277

(3b),

278

hexenenitrile

279

(methylsulfinyl)pentanenitrile

280

acetonitrile (12b), and 1-methoxyindole-3-acetonitrile (13b). Four could be assigned

281

to

282

epithiopentanenitrile

283

epithiohexanenitrile (6cx). EPTs were the main breakdown products formed in the

284

investigated leaves. The total amount of GLS breakdown products comprised 66.9%

285

of EPTs, while ITCs and nitriles represented 22.9% and 10.2%, respectively.

286

Accordingly, EPTs dominated the GLS breakdown products in pak choi (B. rapa ssp.

x

were only tentatively identified compounds. Tentative identification was done

4-pentenyl

ITC ITC

(5a),

5-vinyl-1,3-oxazolidine-2-thione

(4a),

4-

(7a),

5-(methylsulfanyl)pentyl

(8ax),

4-

(4bx),

3-hydroxypentenenitrile

the

(4Pent-CN),

group

of

5-(methylsulfanyl)pentanenitrile

(10bx),

3-phenylpropanenitrile

4,5-epithiopentanenitrile

5,6-epithiohexanenitrile

(5c),

(7bx),

(8bx),

6-(methylsulfanyl)hexanenitrile

EPTs: (4cx),

ITC

(11b),

55-

indole-3-

(3c),

3-hydroxy-4,5-

and

3-hydroxy-5,6-

11



Page 14 of 34

287

chinensis) as well.52 Nevertheless, nitriles were formed in every accession; only one

288

accession (VIR 74) did not form EPTs, and 18 accessions did not form ITCs in

289

detectable amounts. Since the formation of EPTs during GLS hydrolysis requires the

290

specifier protein ESP, presumably all accessions that carried out EPTs possessed

291

ESP activity.53 In accordance with their respective precursor GLS 3c was the main

292

GLS degradation product followed by 3a, 4cx, 5a and 5c (Table 2). This is consistent

293

with our earlier study of the 16 B. rapa turnip accessions.33 Even though ITC 3a was

294

one of the main GLS breakdown products, 3a accumulated in only 21 out of 91

295

accessions. High concentrations of 3a were found in accessions VIR 215, VIR 242

296

and VIR 264 (0.47 to 0.95 µmol g-1 FW). The EPT 3c prevailed in 25 accessions and

297

occurred in relatively high amounts in VIR 337, VIR 241 and VIR 374 (0.83 to 1.3

298

µmol g-1 FW). With 8.12 µmol g-1 FW, the 3c concentration formed from P-143 was

299

highest (Table 2). 4cx predominated in 15 accessions: in VIR 48, VIR 241 and VIR

300

1283 comparatively high concentrations of 4cx could be detected (0.14 to 0.3 µmol g-

301

1

302

(0.16 to 0.33 µmol/g FW), and it was the main GLS breakdown product in VIR 214,

303

VIR 13, and VIR 335. 3c, 5c, and 11b were found in most of the 91 accessions

304

(Table 2). Regarding the five main subspecies, all accessions of B. rapa ssp.

305

chinensis and B. rapa ssp. rapa formed 3c, 11b, and 5c. B. rapa ssp. dichotoma and

306

B. rapa ssp. oleifera f. annua persistently formed 11b, but none of the GLS

307

breakdown products occurred in every accession of B. rapa ssp. pekinensis.

308

Certainly VIR74 exclusively formed nitriles and VIR247 almost formed only nitriles.

309

While ESP favors nitrile formation from non-alkenylic GLSs, this cannot explain the

310

enhanced formation of nitriles from alkenyl-GLS.54 However, in A. thaliana, nitrile

311

specifier proteins (NSPs) were reported to catalyze the formation of simple nitriles.55

FW). The highest 5a concentrations were found in VIR 96, VIR 338 and VIR 241

12


Page 15 of 34


312

Although not yet functionally described in B. rapa, the presence of NSPs could

313

explain favored nitrile formation. In addition, neither a pattern related to the

314

ITC/EPT/CN ratio nor a coherency in the amount of GLS degradation products within

315

one subspecies was found. Similarly to GLS profiles, we found that GLS breakdown

316

product profiles of some accessions displayed a fairly high standard deviation, which

317

could be indicative of a heterogeneous germplasm pool.

318

A Pearson correlation analysis was applied to investigate whether a correlation exists

319

between fresh weight and GLS breakdown product concentration, but revealed no

320

such dependency (r = 0.166, p = 0.117). A Pearson correlation was also applied to

321

evaluate possible relationships within the accumulation of GLS breakdown products

322

and revealed a high correlation between GLS breakdown products derived from the

323

same precursor GLS (Table 3). Accordingly, significant correlations (p ≤ 0.001) were

324

found for 3b and 3c which share 3 as the corresponding intact GLS. In addition, the

325

hydrolysis products of related aliphatic GLSs correlated with each other in positive

326

ways (e.g. 3a and 4cx), probably because their parent GLSs correlated as well.

327

Strong correlations were also detected for GLS breakdown products of GLSs with

328

different precursors for instance 11a (precursor 11) and 5a (precursor 5). In addition

329

to that, some ITCs and EPTs (3a and 4cx, 5a and 3c, 5a and 4cx) which are formed

330

in different breakdown pathways showed high correlations. No consistent negative

331

correlation was seen between ITCs and nitriles or EPTs, since probably the initial

332

GLS correlations had a stronger effect than the differences in ESP activity.

333 334

Taxonomic assessment of GLSs and GLS breakdown product accumulation

335

The B. rapa accessions investigated were previously described taxonomically.38 Most

336

of the 91 B. rapa accessions were assigned to five main subspecies: B. rapa ssp. 13



Page 16 of 34

337

chinensis, B. rapa ssp. dichotoma, B. rapa ssp. oleifera f. annua, B. rapa ssp.

338

pekinensis and B. rapa ssp. rapa. Subspecies of B. rapa can also be distinguished

339

based on their morphological appearance.

340

diversity and genetic relationships of B. rapa accessions using molecular markers, for

341

instance amplified fragment length polymorphisms (AFLP), and simple sequence

342

repeat markers (SSR) but also high-throughput profiling of plant metabolites.

343

Bird et al.57 revealed five subpopulations that reflected morphotype and geographic

344

origin by FastStructure Analysis with Single Nucleotide Polymorphism (SNP).

345

Investigating a collection 168 B. rapa accessions, Del Carpio et al.6 found three

346

groups based on AFLP and SSP on the one hand but four groups based on high

347

throughput profiling of metabolites with LC-MS on the other hand. These groupings

348

rarely reflect the taxonomic relationships and diversity between and within B. rapa

349

subspecies and detailed information on grouping of different B. rapa accessions

350

according to their subspecies is scarce. For breeders it is important to have

351

information about the plant metabolite composition and concentration but also about

352

variances of the metabolite profile within one subspecies for targeted crossing and

353

breeding. Therefore, it was of special interest to evaluate how the main subspecies

354

within the investigated B. rapa accessions would differ with regard to their

355

subspecies-characteristic GLS profile and how similar the GLS profiles of all

356

accessions within one subspecies would be. A discriminant function analysis (DFA)

357

of GLS profiles in the five main subspecies was conducted (Figure 4A). All

358

subspecies differed significantly in terms of their GLS profiles. Standardized

359

coefficients revealed a high contribution of GLSs 6, 5, 1, 15, and 3 to the formation of

360

the subspecies-characteristic GLS profiles. According to the p-values of the squared

361

Mahalanobis distances (distances between group mean values), B. rapa ssp.

56

Recent studies showed the genetic

6, 57, 58

14


Page 17 of 34


362

dichtoma and B. rapa ssp. pekinensis showed the highest distance between their

363

GLS profiles, followed by B. rapa ssp. chinensis and B. rapa ssp. rapa. Contrariwise

364

B. rapa ssp. pekinensis and B. rapa ssp. dichtoma showed a high proximity

365

suggesting a high genetic similarity. Additionally, 23 of 25 accessions (92%) within B.

366

rapa ssp. pekinensis were in agreement with the GLS profile of their particular

367

subspecies. This reveals a highly homogeneous GLS profile within this subspecies.

368

On the other hand the GLS profile within B. rapa ssp. rapa was substantially less

369

homogeneous as only 12 out of 18 accessions (67%) were in agreement with the

370

GLS profile of their particular subspecies. Even though in the present study GLS

371

profiles of the five main subspecies were relatively homogeneous within each

372

subspecies this homogeneity differed between the five main subspecies and thereby

373

could be considered subspecies-characteristic. The DFA of the GLS breakdown

374

product profiles (except 10a, 10bx, 8ax, 13a, 13b) was in agreement with results

375

obtained for GLS profiles. However the revealed significant taxonomic differences of

376

the GLS breakdown product profiles were less distinct compared to the intact GLS

377

profiles (Figure 4B). Compounds 1a and 3c contributed highest to the formation of

378

the subspecies-characteristic GLS breakdown product profiles. Squared Mahalanobis

379

distances revealed the strongest differences between B. rapa ssp. dichotoma and B.

380

rapa ssp. rapa followed by B. rapa ssp. dichtoma and B. rapa ssp. oleifera as well.

381

The proximity of B. rapa ssp. dichtoma, B. rapa oleifera f. annua and B. rapa ssp.

382

pekinensis partially agrees with results obtained for GLS profiles and reinforces the

383

suggested genetic similarity. In contrast to the results obtained for the intact GLSs,

384

the subspecies B. rapa ssp. chinensis and B. rapa ssp. rapa differed significantly in

385

their GLS breakdown product profiles. Consistent to their respective intact GLS

386

profiles, 23 out of 25 accessions (92%) within B. rapa ssp. pekinensis were in 15



Page 18 of 34

387

agreement with the GLS breakdown product profile of their particular subspecies

388

thereby showing a high homogeneity. For B. rapa ssp. oleifera f. annua a weak

389

homogeneity was revealed since only three out of 14 accessions (21%) were in

390

agreement with the GLS breakdown product profile of their particular subspecies. In

391

conclusion the DFA exemplified the similarities and differences of the GLS profiles

392

between all five main subspecies but also between all accessions within one

393

subspecies.

394

Phenotypic variation clearly illustrates genetic diversity, but in order to enlarge the

395

genetic diversity of plant species used in food production, more attention should

396

focus on their biochemical variations. GLSs contribute to this diversity and can be

397

highly beneficial not only for human consumption due to their health promoting

398

effects but also for their potential in biofumigation. This diversity can be found among

399

various B. rapa species whereas the homogeneity of the GLS profile within one

400

subspecies is characteristic for each subspecies. The bioactive effects of GLSs are

401

mainly driven by their breakdown products. While aliphatic and arylaliphatic

402

hydrolysis products can be basically assessed by GC-MS, the analysis of many

403

indole GLS hydrolysis products is difficult due to the high reactivity of especially

404

indole-3-carbinols and the low volatility of these compounds.59 Therefore, several

405

indoles cannot be easily analysed by the standard GC-method. Thus, future

406

investigations should focus on profiling both GLSs and their breakdown products

407

including also non-volatile hydrolysis products.

16


Page 19 of 34


408

Abbreviations:

409

GLS(s),

410

protein(s); NSP, nitrile specifier protein; TFP, thiocyanate-forming protein; EPT(s),

411

epithionitrile(s); ESM, epithiospecifier modifier; DFA, discriminant function analysis;

412

QTL, quantitative trait locus

glucosinolates(s);

ITC(s),

isothiocyanate(s);

ESP(s),

epithiospecifier

413 414

Acknowledgement

415

We thank A. Jankowsky for excellent technical assistance. H.R. Glatt (German

416

Institute of Human Nutrition, Department of Nutritional Toxicology, Potsdam-

417

Rehbrücke, Germany) and S. Rohn (University of Hamburg, Germany) are thanked

418

for providing reference substances.

419 420

Supporting Information description

421 422

Supplemental Figure S1: Examples of leaf phenotypes of the analyzed Brassica rapa

423

accessions (one square = 1x1cm).

424 425

Supplemental Figure S2: Ratio of Nitrile/EPT/ITC in the 91 analyzed Brassica rapa

426

accessions 1, Brassica rapa ssp. chinensis; 2, B. rapa ssp. dichotoma; 3, B.rapa ssp.

427

nipposinica; 4, B. rapa ssp. oleifera f. annua; 5, B. rapa ssp. pekinensis; 6, B.rapa

428

ssp. rapa; 7, B. rapa ssp. sylvestris; 8, B. rapa ssp. trilocularis; 9, stable natural

429

hybrid ssp. chinensis x ssp. chinensis var. rosularis; 10, stable natural hybrid ssp.

430

pekinensis x ssp. chinensis.

431

17



Page 20 of 34

432

Supplemental Table S1: Summary of Brassica rapa accessions analyzed in this

433

study.

434 435

Supplemental Table S2: Absolute glucosinolate (GLS) concentration in leaves of the

436

Brassica rapa accessions (Results are presented as means ± SD of three technical

437

replicates, consisting of three plants each, in µmol/g fresh weight). Errors denote

438

standard deviation. Error bars show the standard deviation of three replicates.

439

Compound numbers are presented in Table 1.

440 441

Supplemental Table S3: Absolute concentration of GLS breakdown products in

442

leaves of the Brassica rapa accessions (Results are presented as means ± SD of

443

three technical replicates, consisting of three plants each, in µmol/g fresh weight).

444

Errors denote standard deviation. Error bars show the standard deviation of three

445

replicates. Compound numbers are presented in Table 1.

446 447

Supplemental Table S4: Pearson Product Moment Correlation of 1) fresh

448

weight/glucosinolate concentration and 2) fresh weight/glucosinolate breakdown

449

product concentration.

450 451

Supplemental Table S5: Discriminant function analysis (DFA) of GLS profiles of the

452

five main subspecies (B. rapa ssp. chinensis, B. rapa ssp. dichotoma, B. rapa ssp.

453

oleifera f. annua, B. rapa ssp. pekinensis, B. rapa ssp. rapa) in the Brassica rapa

454

collection.

18


Page 21 of 34


455

Supplemental Table S6: Discriminant function analysis (DFA) of GLS breakdown

456

product profiles of the five main subspecies (B. rapa ssp. chinensis, B. rapa ssp.

457

dichotoma, B. rapa ssp. oleifera f. annua, B. rapa ssp. pekinensis, B. rapa ssp. rapa)

458

in the Brassica rapa collection.

459

Supplemental Table S7: GC-MS spectra including retention time of all GLS

460

breakdown products identified in the 91 Brassica rapa accessions.

461

19



Page 22 of 34

462

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463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509

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35. Del Carpio, D. P.; Basnet, R. K.; Arends, D.; Lin, K.; De Vos, R. C.; Muth, D.; Kodde, J.; Boutilier, K.; Bucher, J.; Wang, X., Regulatory network of secondary metabolism in Brassica rapa: insight into the glucosinolate pathway. PloS one 2014, 9, e107123. 36. Geilfus, C.-M.; Hasler, K.; Witzel, K.; Gerendás, J.; Mühling, K. H., Interactive effects of genotype and N/S-supply on glucosinolates and glucosinolate breakdown products in Chinese cabbage (Brassica rapa L. ssp. pekinensis). J. Appl. Bot. Food Qual. 2016, 89. 37. Zhao, J. J.; Artemyeva, A.; Del Carpio, D. P.; Basnet, R. K.; Zhang, N. W.; Gao, J.; Li, F.; Bucher, J.; Wang, X. W.; Visser, R. G. F.; Bonnema, G., Design of a Brassica rapa core collection for association mapping studies. Genome 2010, 53, 884-898. 38. Artemyeva, A. M.; Solov'eva, A. E.; Berensen, F. A.; Kocherina, N. V.; Chesnokov, Y. V., Ecological and genetic evaluation of morphological and biochemical characters of quality in Brassica rapa L. accessions from VIR collection. Agric. Biol. 2017, 52, 129-142. 39. Witzel, K.; Hanschen, F. S.; Klopsch, R.; Ruppel, S.; Schreiner, M.; Grosch, R., Verticillium longisporum infection induces organ-specific glucosinolate degradation in Arabidopsis thaliana. Front Plant Sci 2015, 6, 508. 40. EN ISO 9167-1: 1995-09: Rapssamen - Bestimmung des Glucosinolatgehaltes - Teil 1: HPLC-Verfahren (ISO 9167-1: 1992). 41. Zimmermann, N. S.; Gerendás, J.; Krumbein, A., Identification of desulphoglucosinolates in Brassicaceae by LC/MS/MS: Comparison of ESI and atmospheric pressure chemical ionisation‐MS. Mol. Nutr. Food Res. 2007, 51, 15371546. 42. Linsinger, T.; Kristiansen, N.; Beloufa, N.; Schimmel, H.; Pauwels, J., BCR information. Reference Materials 2001. 43. Hanschen, F. S.; Klopsch, R.; Oliviero, T.; Schreiner, M.; Verkerk, R.; Dekker, M., Optimizing isothiocyanate formation during enzymatic glucosinolate breakdown by adjusting pH value, temperature and dilution in Brassica vegetables and Arabidopsis thaliana. Sci. Rep. 2017, 7. 44. Brown, P. D.; Tokuhisa, J. G.; Reichelt, M.; Gershenzon, J., Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochem. 2003, 62, 471-481. 45. Lee, M. K.; Chun, J. H.; Byeon, D. H.; Chung, S. O.; Park, S. U.; Park, S.; Arasu, M. V.; Al-Dhabi, N. A.; Lim, Y. P.; Kim, S. J., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp pekinensis) and their antioxidant activity. LWT - Food Sci. Techno. 2014, 58, 93-101. 46. Zang, Y. X.; Kim, H. U.; Kim, J. A.; Lim, M. H.; Jin, M.; Lee, S. C.; Kwon, S. J.; Lee, S. I.; Hong, J. K.; Park, T. H.; Mun, J. H.; Seol, Y. J.; Hong, S. B.; Park, B. S., Genome-wide identification of glucosinolate synthesis genes in Brassica rapa. FEBS Journal 2009, 276. 47. Kliebenstein, D. J.; Lambrix, V. M.; Reichelt, M.; Gershenzon, J.; MitchellOlds, T., Gene duplication in the diversification of secondary metabolism: tandem 2oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis. Plant Cell 2001, 13, 681-693. 48. Wang, H.; Wu, J.; Sun, S.; Liu, B.; Cheng, F.; Sun, R.; Wang, X., Glucosinolate biosynthetic genes in Brassica rapa. Gene 2011, 487, 135-142. 22


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643 644

23



Page 26 of 34

Table 1: Nomenclature of glucosinolates (GLS) and their corresponding GLS breakdown products detected in Brassica rapa accessions (n.d. = not detected, - = not formed from precursor GLS). Glucosinolates

Corresponding breakdown products Isothiocyanate

Nitrile

Epithionitrile

1

Name 1-methylpropyl

Abbreviation 1a

Name sec-butyl ITC

Abbreviation 1b

Name 3-methylpentanenitrile

Abbreviation -

Name -

2

2-propenyl

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

3

3-butenyl

3a

3-butenyl ITC

3b

4-pentenenitrile

3c

4

4a

3-hydroxypentenenitrile

4c

5a

5-vinyl-1,3oxazolidine-2-thione 4-pentenyl ITC

4b

5

2-(R)-2-hydroxy-3butenyl 4-pentenyl

5b

5-hexenenitrile

5c

4,5epithiopentanenitrile 3-hydroxy-4,5epithiopentanenitrile 5,6-epithiohexanenitrile

6

2-hydroxy-4-pentenyl

n.d.

n.d.

n.d.

n.d.

6c

7

4-(methylsulfanyl)butyl

7a

7b

5-(methylsulfanyl)pentyl

8a

-

-

9

3-(methylsulfinyl)propyl

n.d.

5-(methylsulfanyl) pentanenitrile 6-(methylsulfanyl) hexanenitrile n.d.

-

8

4-(methylsulfanyl) butyl ITC 5-(methylsulfanyl) pentyl ITC n.d.

3-hydroxy-5,6epithiohexanenitrile -

-

-

10

4-(methylsulfinyl)butyl

10a

11

2-phenylethyl

12 13 14 15

8b n.d. 10b

11a

4-(methylsulfinyl) butyl ITC 2-phenylethyl ITC

-

-

11b

5-(methylsulfinyl)pentanenitrile 3-phenylpropanenitrile

-

-

3-indolylmethyl

n.d.

n.d.

12b

indole-3-acetonitrile

-

-

1-methoxy-3indolylmethyl 4-methoxy-3indolylmethyl 4-hydroxy-3indolylmethyl

13a

13b

-

n.d.

1-methoxyindole-3acetonitrile n.d.

-

n.d.

1-methoxyindole-3carbinol n.d.

-

-

n.d.

n.d.

n.d.

n.d.

-

-


Page 27 of 34

645 646 647 648


Table 2: Pearson correlation coefficients among all identified glucosinolates in Brassica rapa accessions. Asterisks and blue color indicate significant correlations at p < 0.05 (*), 0.01 (**), 0.001 (***).Compounds marked with (x) were only tentatively identified (by comparing their mass spectra and retention time with the literature). Compound numbers are presented in Table 1. 2 x

1 2 3 4 5 6 7 8 9

-

3

4

5

6

7

8

9

10

11

I12

13

14

15

-0.05

0.14

0.42*

0.02

0.32

0.66

0.16

0.83

0.08

0.15

-0.05

-0.15

0.10

-0.76

0.92

-0.34

0.00

-

-

0.00

-0.89

0.57

0.86

0.89

0.70

0.80

-0.05

0.37***

-0.22

0.36

-0.14

-0.12

0.88***

0.28**

-0.13

-0.02

0.20

-0.15

0.25*

0.51***

0.09

0.72*

0.34

0.58

0.28**

0.31**

0.15

0.19

0.28**

0.32**

0.22

0.55

-0.07

0.05

0.58***

0.14

-0.01

0.15

0.21*

0.29

0.58

0.13

0.48

0.24*

0.61***

-0.04

0.04

0.50***

0.24

0.26

0.92

0.33

0.07

-0.11

0.55

0.19

0.49

0.10**

0.62*

0.29

-0.13

0.65*

0.86***

-1.00

-0.06

0.34

0.53**

0.40*

0.23

0.33

0.18

0.17

0.40

0.16

0.12

0.14

0.25*

0.27**

0.01

0.20*

0.58***

0.09

-0.12

10 11 12 13

0.26

14

649 650



0.00

0.01

0.90*

1.00 ***

1.00

0.21

0.09

0.72

0.04

0.28

0.63

0.80 0.46*

5a

0.35

0.99

1.00

0.18

-

0.57

0.24 0.25 * 0.10

0.14 0.28

5c x

0.98 0.18 1.00 0.29 0.17 0.51 0.04

1.00 0.79

x

10a 10b

x

-

-

1.00

-

1.00

1.00

0.00

-

0.88

0.35

0.79

0.00

-

0.12 0.39 0.54

0.62 ** 0.59 *** 0.77 ***

-

-

0.00

0.69

1.00

-

-

0.00

0.00

0.00

-

-

-

0.00

0.00

0.00

0.00

0.00

0.00

0.18

0.56

0.48

0.05

1.00

1.00

0.78

1.00

-

-

-

0.00

-

-

-

0.69

-

-

-

-

1.00

-

1.00

0.19

0.07

0.00

0.69

0.46 * 0.80 ***

0.57 *** 0.31 * 0.57 ***

0.00

0.00

0.00

0.29

0.08

1.00

0.53

0.85

1.00

-

-

1.00

-

1.00

1.00

1.00

0.99

1.00

-1.00

-

0.00

-

-

0.00

-

-

-

-

x

11a

1.00 1.00 0.00

0.98 0.40 0.50 -

0.17

0.17 0.41 *** 0.10

1.00 0.24 0.09 0.55 0.49 0.13 0.13 0.45 *

13b

0.00

0.03

-

0.00

x

8b

0.35

-

-

5b 8a

0.65

x

-

8b

5b

10bx 0.00

0.29

0.25

7b

-

8ax

-

1.00

-

7a

10a

0.09

-

7bx

0.35 0.75 ***

x

0.30

7a

0.06

6cx

4c

-0.63

1.00 0.76 * 0.63 ** 0.41 * 0.55 *** 0.88 *

13a

x

4a

3c

3b

0.07

0.20

12b

4a

6c

0.16

-0.31

11b

3c

4c

0.06

0.70 ***

3b

4b

-0.01

1.00 1.00

11a

3a

0.06

5c

0.19

x

1.00*

5a

1b

0.00

x

0.07

4bx

1a

3a

Table 3: Pearson correlation coefficients among all identified glucosinolate breakdown products in Brassica rapa accessions. Asterisks and blue color indicate significant correlations at p < 0.05 (*), 0.01 (**), 0.001 (***). Compounds marked with (x) were only tentatively identified (by comparing their EI mass spectra with the literature). Compound numbers are presented in Table 1. 1bx

651 652 653 654

Page 28 of 34

0.05

0.01

0.00

0.00

-

0.47

0.64

0.53

0.00

-

-

0.10

0.32

0.81

0.00

-

-

0.00

1.00

1.00

-

-

-

-

-

1.00

1.00

0.00

0.00

-

-

0.00

-

0.70

0.00

-

-

1.00

1.00

1.00

-

-

-

0.25

0.66

1.00

-

-

0.25

0.21

0.00

0.00

26


Page 29 of 34


11b

0.12

12b 13a

0.00

0.00

0.00

-

655 656 657 658 659 660 661 662

27



663

Figures

664

Figure 1: Schematic representation of enzymatic glucosinolate (GLS) hydrolysis,

665

representing the hydrolysis of the main Brassica rapa GLS, 3-butenyl GLS (3), to 3-

666

butenyl ITC (3a), 4-pentenenitrile (3b), or 4,5-epithiopentanenitrile (3c).

667 668


Page 30 of 34

Page 31 of 34


669

Figure 2: The main glucosinolates (GLS) in leaves of the Brassica rapa collection.

670

Values represent means of three replicates, comprising two plants each. GLS are

671

shaded in colors similar to their corresponding breakdown products (see Fig. 3);

672

compound numbers are presented in Table 1. Accessions were grouped according to

673

their taxonomy (1, B. rapa ssp. chinensis; 2, B. rapa ssp. dichotoma; 3, B. rapa ssp.

674

nipposinica; 4, B. rapa ssp. oleifera f. annua; 5, B. rapa ssp. pekinensis; 6, B. rapa

675

ssp. rapa; 7, B. rapa ssp. sylvestris; 8, B. rapa ssp. trilocularis; 9, stable natural

676

hybrid B. rapa ssp. chinensis x B. rapa ssp. chinensis var. rosularis; 10, stable

677

natural hybrid B. rapa ssp. pekinensis x B. rapa ssp. chinensis). Error bars show

678

standard deviation of three biological replicates comprising of two plants.

679

29



Page 32 of 34

680

Figure 3: The main glucosinolate (GLS) breakdown products in leaves of the

681

Brassica rapa collection. Values represent means of three replicates, comprising

682

three plants each. Breakdown products are shaded in colors similar to their intact

683

precursor (see Fig. 2); compound numbers are presented in Table 1. Accessions

684

were grouped according to their taxonomy (1, B. rapa ssp. chinensis; 2, B. rapa ssp.

685

dichotoma; 3, B. rapa ssp. nipposinica; 4, B. rapa ssp. oleifera f. annua; 5, B. rapa

686

ssp. pekinensis; 6, B. rapa ssp. rapa; 7, B. rapa ssp. sylvestris; 8, B. rapa ssp.

687

trilocularis; 9, stable natural hybrid B. rapa ssp. chinensis x B. rapa ssp. chinensis

688

var. rosularis; 10, stable natural hybrid B. rapa ssp. pekinensis x B. rapa ssp.

689

chinensis). Error bars show standard deviation of three biological replicates

690

comprising of two plants. 691

30


Page 33 of 34


692

Figure 4: Discriminant function analysis (DFA) of glucosinolate (GLS) profiles (A) and

693

GLS breakdown product profiles (B) of the five main subspecies (Brassica rapa ssp.

694

chinensis, B. rapa ssp. dichotoma, B. rapa ssp. oleifera f. annua, B. rapa ssp.

695

pekinensis, B. rapa ssp. rapa). Ellipses indicate the 90% confidence interval.

696

B. rapa ssp. chinensis

B. rapa ssp. dichtoma

697

B. rapa ssp. pekinensis

B. rapa ssp. rapa

B. rapa ssp. oleifera f. annua

698 699

A

700 701 702 703 704 705 706 707 708 709 710 711

B

712 713 714 715 716 717 718 719

31



720

Page 34 of 34

TOC graphic

721

722 723 724

32


Genotypic variation of glucosinolates and their breakdown products in

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