Effects of selenium supplementation on glucosinolate biosynthesis in

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Bioactive Constituents, Metabolites, and Functions

Effects of selenium supplementation on glucosinolate biosynthesis in broccoli Ming Tian, Yong Yang, Fabricio William Avila, Tara Fish, Hui Yuan, Maixia Hui, Siyi Pan, Ted Thanhauser, and Li Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03396 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

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Effects of selenium supplementation on glucosinolate biosynthesis in broccoli

2 3

Ming Tiana,b, Yong Yanga, Fabricio William Ávilaa,c, Tara Fisha, Hui Yuana,e, Maixia Huia,d,

4

Siyi Panb, Theodore W Thannhauser a, Li Lia,e,*

5 6

a

7

New York 14853, USA;

8

b

9

Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China;

Robert W Holley Center for Agriculture and Health, USDA-ARS, Cornell University, Ithaca,

Key Laboratory of Environment Correlative Dietology, Ministry of Education, College of

10

c

State University of Mid West, UNICENTRO, Paraná, Brazil;

11

d

College of Horticulture, Northwest A & F University, Yangling, 712100, China;

12

e

13

University, Ithaca, New York 14853, USA

Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell

14 15 16

* Corresponding author:

17

Tel: 1-607-255-5708; Email: [email protected]

18

1 ACS Paragon Plus Environment


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ABSTRACT

20 21

Selenium (Se) enriched broccoli adds health beneficial selenium-containing compounds, but

22

may contain reduced amounts of chemopreventive glucosinolates. To investigate the basis by

23

which Se treatment influences glucosinolate levels, we treated two broccoli cultivars with 25

24

µM Na2SeO4. We found that the Se supplementation suppressed the accumulation of total

25

glucosinolates, particularly glucoraphanin, the direct precursor for a potent anticancer

26

compound, in broccoli florets and leaves. We showed that the suppression was not associated

27

with plant sulfur nutrition. The levels of glucosinolate precursors methionine and

28

phenylalanine as well as the expression of genes in the glucosinolate biosynthesis were

29

greatly decreased following Se supplementation. Comparative proteomic analysis identified

30

proteins in multiple metabolic and cellular processes that were greatly affected and detected

31

an enzyme affecting methionine biosynthesis that was reduced in Se biofortified broccoli.

32

These results indicate that the Se conferred glucosinolate reduction is associated with its

33

negative effects on precursor amino acid biosynthesis and glucosinolate biosynthetic gene

34

expression, and provide information for a better understanding of glucosinolate accumulation

35

in response to Se supplement in broccoli.

36 37

KEYWORDS: Broccoli, selenium, glucosinolate, gene expression, amino acid, proteomics

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39

INTRODUCTION

40 41

Glucosinolates, a group of secondary metabolites found in almost all plants of the order

42

Brassicales, have received special attention due to their importance in human health and plant

43

defense.

44

β-thioglucoside, a sulfonated oxime moiety, and a variable aglycone side chain derived from

45

an amino acid. Based on the chemical structures of the precursor amino acids, glucosinolates

46

are classified into three groups: aliphatic glucosinolates from alanine, leucine, isoleucine,

47

valine, and methionine; benzenic glucosinolates from phenylalanine and tyrosine; and indolic

48

glucosinolates from tryptophan 1. The major glucosinolates of Arabidopsis thaliana are

49

derived from methionine, phenylalanine, and tryptophan 2. Glucosinolates are synthesized via

50

three independent stages: (i) chain elongation of selected precursor amino acids, (ii)

51

formation of the core glucosinolate structure, and (iii) secondary modifications of the amino

52

acid side chain 3. Many genes in the glucosinolate biosynthetic pathway and a number of

53

transcription factors have been identified (Figure 1). While glucosinolate enzymatic

54

degradation products account for their bioactivities on herbivores and pathogens in plants 4,

55

some enzymatic hydrolysis compounds, such as sulforaphane and indole-3-carbinol, are

56

known to possess anticancer, antidiabetic, antimicrobial, and cholesterol lowing properties to

57

humans 5–7.

Glucosinolates

as

sulfur-

and

nitrogen-containing

compounds

comprise

58

Selenium (Se) is an essential micronutrient for animals and humans, and has other

59

health benefits including being a cancer preventive agent. Plant foods contain various forms

60

of Se including inorganic selenate and selenite, selenoamino acids selenocysteine and

61

selenomethionine,

62

γ-glutamyl-Se-methylselenocysteine (GGSeMSCys) 8. While different forms of Se provide

63

different levels of protection against cancer, monomethylated SeMSCys and GGSeMSCys

and

monomethylated

Se-methylselenocysteine


(SeMSCys)

and


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9,10

64

offer superior cancer chemopreventive activities

65

and selenite that induce oxidative stress and produce malformed selenoproteins to affect

66

protein normal functions when in excess, the monomethylated forms of Se such as SeMSCys

67

can’t be integrated into proteins and is efficiently converted to non-toxic, chemopreventive

68

methylselenol in body 11. SeMSCys was found to be the major selenocompound in selenium

69

enriched foods such as garlic, onions, broccoli, and wild leeks 8.

70

. In addition, unlike inorganic selenate

Selenium is a sulfur (S) analog and shares the S uptake and assimilation pathways in 12–14

71

plants

72

number of plants

73

nutrition in plants to affect plant metabolism 18–20.

. While Se fertilization at low dosages has been shown to enhance S levels in a 15–17

, Se application at higher dosages exerts antagonistic effect on S

74

Broccoli (Brassica oleracea L. var. italica) has long been included in human diet and

75

represents one of the most nutrient-dense and popular vegetables. It is rich in multiple

76

nutrients (i.e. vitamins and minerals) and many health beneficial compounds, including

77

glucosinolates

78

23,24

79

respectively

80

amounts of bioactive SeMSCys when grown in Se-containing soils or media

81

studies reveal that broccoli also produces selenoglucosinolates following Se fertilization 26,27,

82

which have implications with more potent anticancer activity

83

investigated for simultaneous production of glucosinolates and organoselenium compounds,

84

such as SeMSCys, to high levels as a functional food for cancer prevention. Selenium

85

supplement at low dosages has been shown to exert minimal effects on glucosinolate

86

accumulation

87

SeMSCys in broccoli 19. While the beneficial Se-containing compounds can be significantly

88

increased with increased Se dosages

21,22

. Broccoli contains high abundance of glucoraphanin and glucobrassicin

. Their hydrolysis leads to the production of sulphoraphane and indole-3-carbinol, 6,25

. In addition, broccoli as a Se-accumulating crop synthesizes significant 8,19

. Recent

28

. Broccoli has been

29–31

. However, the low dosages also lead to low production of bioactive

19,23

, several studies have shown that Se treatments 4

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reduce glucosinolate levels in shoots and florets of broccoli 23,32,33. The negative effect of Se

90

treatments on glucosinolate content was also observed in other Brassica species

91

is thought to be due to the antagonistic effect of Se on plant S nutrition, which directly

92

influences glucosinolate metabolism. However, the basis by which glucosinolate levels are

93

affected by Se treatment is not very clear.

16,34,35

. This

94

In this study, we examined the effects of Se supplied as 25 µM Na2SeO4 on total and

95

individual glucosinolate levels in sprouts, young leaves and florets of two broccoli cultivars.

96

The total levels of S and amino acids related to glucosinolate biosynthesis as well as the

97

expression of genes involved in the glucosinolate biosynthetic pathway were evaluated. The

98

global proteome changes in florets in response to selenate treatment were also investigated to

99

examine the metabolic and cellular processes affected, and gain a better understanding of the

100

key factors that affect glucosinolate levels following Se supplement in broccoli.

101 102

MATERIALS AND METHODS

103

Plant materials and treatments. Broccoli (Brassica oleracea L. var. italica) seeds of

104

two commercial cultivars Diplomat and Gypsy were obtained from Harris Seeds (Rochester,

105

NY) and used in this study. For experiments with sprouts, the seeds were sown on two sheets

106

of filter paper (3 mm, Whatman) soaked with treatment solutions in Magenta boxes, and

107

germinated in a growth chamber with a photoperiod of 16 h of light and 8 h of dark at 22°C.

108

Treatment solutions were either Milli-Q water (control) or 25 µM sodium selenate (Na2SeO4).

109

The Se treatment was chosen because we have previously shown that selenate is better than

110

selenite to induce Se and particularly SeMSCys accumulation and the dosage of 25 µM

111

Na2SeO4 does not affect broccoli plant growth

112

spouts were harvested, washed extensively with Milli-Q water, and frozen in liquid nitrogen,

113

followed by either lyophilizing dry or storing at -80 °C.

19,21,23,36

. After 7 days of germination, the



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114

For experiments with broccoli young leaves, seeds were germinated in rolls of

115

moistened filter papers. The young seedlings were grown hydroponically in pots containing

116

Hoagland solution in a greenhouse as described previously 17. Following one-week of growth

117

in the nutrition solution, half of the seedlings were exposed to 25 µM Na2SeO4 and the other

118

half were maintained in the nutrition solution as controls. Two weeks after the treatments, the

119

young leaf samples were harvested and then either freeze-dried or stored at -80 °C.

120

For experiments with broccoli florets, seeds were sown in soil and grown in a

121

greenhouse with a photoperiod of 14/10 h of light/dark at 24°C. When plants just initiated

122

floral primordia, six applications (twice per week for three weeks) of 100 mL of 1.5 mM

123

Na2SeO4 solution were applied to each pot filled with 6 dm3 of soil mix (Metro-Mix 360, Sun

124

Gro Horticulture), resulting in a final dosage of 25 µM Na2SeO4 in each application. When

125

the florets were fully formed and at market harvest maturity, florets were harvested and then

126

either freeze-dried or stored at -80°C.

127

Analysis of S and Se levels by ICP. Total S and Se levels in the sprouts, young

128

leaves, and florets were determined using an inductively coupled plasma (ICP) trace analyzer

129

emission spectrometer (model ICAP 61E trace analyzer, Thermo Electron, San Jose, CA)

130

essentially as described previously

131

were acid-digested in 2.0 mL of H3NO3 with 2.0 mL of HClO4 at 120 ˚C for 1 h and then at

132

220 ˚C until HClO4 fumes were observed. Digested samples were solubilized to a final

133

volume of 20 mL in water before analysis. Each sample was analyzed in triplicate.

134

37

. In brief, approximately 200 mg freeze-dried tissues

Analysis of glucosinolates by UPLC-MS/MS. Total glucosinolates were extracted 23,36,38

135

and analyzed essentially following the protocol described previously

136

freeze-dried samples of approximately 20 mg were extracted in 1.0 mL of 80% MeOH at

137

80 °C and desulfated in a DEAE Sephadex A-25 column with the addition of sulfatase

138

enzyme. The desulfoglucosinolates were eluted from the column, dried, and reconstituted in 6 ACS Paragon Plus Environment

. Briefly,

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0.1% formic acid. Analysis of the reconstituted samples was carried out on an Acquity UPLC

140

(Waters, Milford, MA) using an HSS T3 column (2.5 µm particle, 2.1 x 150 mm column).

141

The glucosinolates were eluted in a liner gradient with a mobile phase consisting of solvent A

142

(0.1% aqueous formic acid in water) and solvent B (0.1% FA in acetonitrile) in a total of 7.5

143

minutes. All glucosinolates were quantitated based on UV absorbance at 227 nm from an

144

Acquity PDA detector (Waters).

145

The elution flow was further directed to a Xevo G2 Q-ToF MS (Waters) to verify the 23

146

identity of the individual glucosinolates via m/z

. The m/z range from 50-1200 was

147

monitored with 1 scan every 2 s. A Waters Lockspray ESI source was used with capillary

148

voltage at 2.5 kV and sampling cone at 2 V. All instruments were controlled by Waters

149

MassLynx software, V4.1 SCN 862.

150

Analysis of free amino acid involved in glucosinolate biosynthesis. Levels of free

151

amino acids were analyzed according to the method described previously with some

152

modifications 39. Free amino acids were extracted overnight from freeze-dried tissues (25 mg)

153

at 4 °C in 50 mM HCl (20:1, v/w). The mixtures were centrifuged at 12,000 g for 15 min and

154

the extracted amino acids in the supernatants were tagged AccQ·Tag using the AccQ·Tag

155

Ultra UPLC derivatization kit according to the manufacturer’s instruction (Waters).

156

Derivatized amino acids were analyzed on an Acquity UPLC™ system (Waters) using an

157

AccQ.Tag Ultra column (100 mm × 2.1 mm). Free amino acids were identified based on

158

co-elution with Pierce Amino Acid Standard H (Thermo Scientific). The total content was

159

calculated based on peak areas and calibration curves generated with commercial standards.

160

RNA extraction, reverse transcription, and quantitative PCR analysis. Total

161

RNA from 0.1 g of leaves and florets were extracted using TRIzol according to the

162

manufacturer’s protocol (Life Technologist) and reverse-transcribed into cDNA using

163

Superscript III reverse transcriptase (Invitrogen). A quantitative reverse transcription 7 ACS Paragon Plus Environment


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164

polymerase chain reaction (qRT-PCR) was performed using the SYBR Green Universal

165

Master Mix (Applied Biosystems) in an ABI 7500 Real-Time PCR system as described

166

previously

167

of all gene expression was performed with at least two technical trials for each of the three

168

biological replicates.

169

40

. The gene-specific primers used are listed in Supplemental Table S1. Analysis

Proteomics analysis. Proteins from the Na2SeO4 treated and untreated florets were 41

170

extracted using a phenol extraction method

171

biological replicates were included in the analysis. Aliquots of proteins from each sample

172

were denatured, reduced and blocked at the cysteines with S-Methyl methanethiosulfonate

173

(MMTS), and digested following the protocol described by Yang et al

174

peptides were labeled with Tandem Mass Tag™ (TMT) 10-plex reagents following the

175

manufacturer’s recommended protocol (ThermoFisher Scientific). After labeling check, the

176

six samples (2 treatments x 3 biological replicates) were pooled, and subjected to cation

177

exchange chromatography using Mixed-Mode Anion-eXchange (MCX) Cartridges (Waters,

178

Milford, MA). The eluted tryptic peptides were evaporated to dryness and reconstructed in

179

buffer A (20 mM ammonium formate pH 9.5) for the first dimensional high pH reverse phase

180

chromatography.

and quantified by the Bradford assay. Three

38

. The digested

181

The reconstituted samples were loaded onto an XTerra MS C18 column (3.5 µm,

182

2.1x150 mm, Waters) and separated on a Dionex UltiMate 3000 HPLC system equipped with

183

a built-in micro fraction collector and UV detector (Sunnyvale, CA). Liquid chromatography

184

(LC) was carried out using a gradient from 10-45% of buffer B (80% acetonitrile/20% 20

185

mM NH4FA) in 30 minutes at a flow rate of 200 µL/min. Forty-eight fractions were collected

186

and pooled into 16 fractions based on the UV absorbance at 214 nm and with multiple

187

fraction concatenation strategy. All of the fractions were dried and reconstituted for

188

nanoLC-MS/MS analysis. 8 ACS Paragon Plus Environment

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Quantitative LC-MS/MS analysis was carried out using an Orbitrap Fusion

190

(Thermo-Fisher Scientific, San Jose, CA) mass spectrometer equipped with a nanospray Flex

191

Ion Source using high energy collision dissociation (HCD). Peptides were concentrated and

192

desalted on a PepMap C-18 RP nano trap column (5 µm, 100 µm × 20 mm) and then

193

separated on a PepMap C-18 RP nano column (3 µm, 75µm x 25cm) using a 120 min

194

gradient of 5-35% acetonitrile in 0.1% formic acid. MS data was acquired using a

195

data-dependent acquisition (DDA) mode under Xcalibur 3.0 operation software.

196

All MS and MS/MS raw spectra from experiments were processed and searched using

197

Sequest HT software within the Proteome Discoverer 2.2 (Thermo) against Brasscia database

198

containing

199

(http://brassicadb.org/brad/datasets/pub/Genomes/Brassica_oleracea/V1.1/).

200

search settings used were: full trypsin digest with two missed cleavages, fixed modifications

201

of Methylthio for cysteine, and 10-plex TMT modifications on lysine and N-terminal amines.

202

Allowed variable modifications included methionine oxidation. The peptide/fragment mass

203

tolerances were 10 ppm and 0.5 Da, respectively. The TMT 10-plex quantification method

204

within Proteome Discoverer 2.2 was used to calculate the reporter ratios with mass tolerance

205

±10 ppm without applying the isotopic correction factors. Only peptide spectra containing all

206

reporter ions were used for quantitation. Protein ratios are expressed as the median value of

207

the ratios for all quantifiable spectra of the unique peptides pertaining to a specific protein.

208

The differentially expressed proteins were determined based on the following two criteria for

209

identified proteins: (1) p value in Student t-test is less than 0.05, and (2) the ratio of

210

expression between Na2SeO4-treated and control samples was larger than +/- 1.2 fold based

211

on means ± 2 SD of three biological replications for all quantified proteins, which was

212

statistically analyzed using EasyFit software.

45,758

sequence


entries The

default


213 214

MapMan software

43

Page 10 of 26

was used for identifying the functional groups of the

differentially expressed proteins.

215

Statistical analysis. The significant difference among treatments was determined

216

using Duncan’s multiple-range test (P < 0.05 as difference)

217

means + SD of three biological replications for each sample.

44

. All data are shown as the

218 219

RESULTS

220

Selenate decreases glucosinolate levels in leaves and florets. Glucosinolates are a

221

group of the most important secondary metabolites in broccoli. The effects of selenate

222

treatment on total glucosinolate levels in sprouts, young leaves and floret of two broccoli

223

cultivars were examined. As shown in Figure 2A, the glucosinolate content in sprouts of both

224

cultivars Diplomat and Gypsy remained similar when they were treated with and without 25

225

µM Na2SeO4, a concentration with no apparent detriment to the plant growth

226

However, in young leaves and florets of broccoli, supplementation of selenate substantially

227

suppressed total glucosinolate levels (Figure 2B-C). Clearly, broccoli spouts, leaves, and

228

florets responded differently to selenate treatments for glucosinolate accumulation.

19,21,23,36

.

229

The glucosinolate profiles in these broccoli tissues were also analyzed following

230

UPLC separation and MS/MS confirmation of individual glucosinolates. Six major

231

glucosinolates

232

hydroxyglucobrassicin (4-hydroxyindol-3-ylmethyl, HGB), glucobrassicin (indol-3-ylmethyl,

233

GB),

234

(1-methoxyindol-3-ylmethyl, NGB), and glucoerucin (4-methylthiobutyl, GE) (Figure 3A).

235

Glucoraphanin represented the most abundant glucosinolate in sprouts of both broccoli

236

cultivars. Five major glucosinolates were detected in leaves and florets (Figure 3B-C).

237

Neoglucobrassicin was the most abundant glucosinolate in young leaves (Figure 3B). Florets

in

sprouts

methoxyglucobrassicin

were

glucoraphanin

(4-methylsulfinylbutyl,

(4-methoxyindol-3-ylmethyl,


MGB),

GR),

neoglucobrassicin

Page 11 of 26


238

contained high levels of glucoraphanin, glucobrassicin, and neoglucobrassicin (Figure 3C).

239

Among these individual glucosinolates, glucoraphanin and glucoerucin belong to aliphatic

240

glucosinolates, and the others belong to indole glucosinolate.

241

The effects of selenate treatment on individual glucosinolates were examined. In

242

sprouts, selenate treatment did not altered individual glucosinolate levels (Figure 3A).

243

However, the individual glucosinolates in leaf and floret tissues were differently affected by

244

selenate

245

neoglucobrassicin, were suppressed by approximately 50% after selenate treatment in both

246

young leaves and florets (Figure 3B-C), showing that selenate at 25 µM inhibited the

247

accumulation of both aliphatic and indole glucosinolates in these tissues.

treatment.

The

highly abundant glucosinolates, i.e., glucoraphanin and

248

Total S levels in broccoli tissues are not affected by selenate. To see whether the

249

reduced glucosinolate levels were due to a possible decrease in the S content in the broccoli

250

tissues following selenate treatment, we analyzed the total S levels along with Se content in

251

sprouts, young leaves, and florets of the two broccoli cultivars treated with and without 25

252

µM Na2SeO4. ICP analysis revealed that total S levels were similar between control and

253

selenate treated group in these three tissues (Figure 4A), which suggested that selenate

254

treatment at the dosage used did not affect S accumulation and the suppressed glucosinolate

255

levels in the Se-treated samples were not associated with the S levels in these tissues. In

256

addition, the total Se levels in all these three tissues were examined. As expected, the

257

selenate-treated samples accumulated high levels of Se (Figure 4B). The leaf and floret

258

tissues contained much higher levels of Se than sprouts with about 4-5 folds difference.

259

Selenate decreases the levels of amino acids involved in glucosinolate synthesis in

260

leaves and florets. To see whether the glucosinolate-related amino acid levels were affected

261

by selenate treatment, the free amino acids in sprouts, leaves and florets were analyzed by

262

UPLC. The total amino acid levels of both cultivars were not significantly affected by 11 ACS Paragon Plus Environment


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selenate treatment (Figure 5A). Detailed analysis of individual amino acids involved in

264

glucosinolate biosynthesis revealed that the levels of methionine and phenylalanine were

265

greatly decreased by selenate treatment in young leaves and florets of both broccoli cultivars

266

(Figure 5B-C). In contrast, while the tryptophan content was not available, no significant

267

changes were noticed following selenate treatment for the levels of tyrosine (Figure 5D), as

268

well as alanine, valine, isoleucine, and leucine in the broccoli tissues (Supplemental Figure

269

S1).

270

Expression of genes involved in glucosinolate synthesis. To see whether selenate

271

treatment also affected glucosinolate biosynthetic and regulatory gene expression, we

272

examined the transcript levels of some related genes in young leaves and florets. These genes

273

included MYB28 and MYB34 that regulate aliphatic and indole glucosinolate synthesis,

274

respectively, BCAT4 and MAM1 involved in the chain elongation, CYP79B2, CYP79F1,

275

CYP83B1 and CYP83A1 in the formation of core glucosinolate structure, and UGT74B1 and

276

FMO2 for secondary modifications

277

significantly suppressed by 25 µM Na2SeO4 treatment in young leaves (Figure 6). Similarly,

278

the transcript levels of most genes examined were affected in florets (Figure 7). Selenate

279

exposure dramatically downregulated the MYB transcription factors and affected the

280

biosynthetic pathway genes examined, suggesting an inhibitory effect of selenate at the

281

dosage on the genes involved in the synthesis of glucosinolates.

3

(Figure 1). The expression of all genes examined was

282

Global proteome changes contribute to the selenate-suppressed glucosinolate

283

levels. To investigate the effect of Se on glucosinolate biosynthesis at a global proteome level,

284

a TMA-based isobaric labeling technology was used to quantitatively compare the proteomes

285

of broccoli florets with and without selenate treatments. A total of 10,693 proteins were

286

identified and 9,303 of them were quantified. Among these quantified proteins, 338 proteins

287

showed differential expression using the cutoff criteria of >1.2 folds in three biological 12 ACS Paragon Plus Environment

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replicates (Supplemental Table S2). These differentially expressed proteins included 81

289

upregulated and 257 downregulated ones. Noticeably, none of the enzyme proteins involved

290

in the glucosinolate biosynthesis pathway were identified among the differentially expressed

291

proteins (Supplemental Table S2).

292

To examine the cellular processes affected by selenate in broccoli florets, the 43

293

differentially expressed proteins were categorized into functional groups using MapMan

.

294

Apart from the group without an assigned function, the most abundant functional groups are

295

those involved in protein (BIN 29), stress (BIN 20), and MISC (BIN 26), followed by RNA

296

(BIN 27), signaling (BIN 30), secondary metabolism (BIN 16), and transport (BIN 34)

297

(Figure 8).

298

Selenium treatment down-regulated a large number of proteins in the functional group

299

of protein (BIN29) (Supplemental Table S3). Among these proteins, many in the

300

posttranslational modification and degradation were suppressed by selenate, showing the

301

negative effect of Se supplementation on protein modification and metabolism in the florets.

302

In contrast, high numbers of ribosomal proteins were up-regulated. Interestingly, Se treatment

303

down-regulated a large number of stress (BIN 20) responsive proteins including heat shock

304

proteins and those in response to biotic and abiotic stresses. In the functional group of RNA

305

(BIN 27), over half of the down-regulated proteins are those involved in regulation of

306

transcription. A large number of proteins associated with RNA processing were also

307

negatively affected following selenate treatment. Overrepresented leucine rich repeat kinase

308

signaling proteins, flavonoid and lignin biosynthesis, and ABC transporters in the signaling

309

(BIN 30), secondary metabolism (BIN 16), and transport (BIN 34) functional groups,

310

respectively, were down-regulated when supplied with 25 µM of selenate (Supplemental

311

Table S3). The data indicates a wide impact of Se on the cellular and metabolic processes in

312

broccoli florets. 13 ACS Paragon Plus Environment


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313

In addition, an enzyme protein involved in sulfur assimilation and cysteine

314

biosynthesis, i.e, serine O-acetyltransferase, in amino acid metabolism (BIN 11) was

315

down-regulated by selenate treatment (Supplemental Table S3). Cysteine is a precursor for

316

methionine biosynthesis. The reduced expression of serine O-acetyltransferase was consistent

317

with the observed reduction of methionine in florets following selenate treatment (Figure 5B),

318

supporting the notion that the effect of selenate treatment on glucosinolates was associated

319

with the suppression of the related amino acid synthesis.

320 321

DISCUSSION

322

Selenium supplement in Brassica crops provides an effective approach to supply

323

chemopreventive Se compounds

324

impact on S nutrition and metabolites 12–14. Glucosinolates are sulfur-containing metabolites,

325

and their accumulation has been shown to be affected by Se treatments

326

investigated how selenate supplementation affected glucosinolate content in two broccoli

327

cultivars. We showed that Se at the dosage with no apparent detriment to the plant growth

328

reduced glucosinolate levels. The effect was likely due to the negative roles of Se in

329

suppressing the related amino acid synthesis and in affecting glucosinolate pathway gene

330

expression as well as other cellular processes, but not specifically the plant S nutrition.

45,46

. However, as an S analog, Se can impose negative

16,19,23,33,34

. Here we

331

The glucosinolate accumulation in the broccoli tissues was differently affected by Se.

332

Selenate treatment at 25 µM did not affect the total and individual glucosinolates in sprouts

333

of the two broccoli cultivars (Figure 2A and Figure 3A). This was likely because the

334

glucosinolates in sprouts were mainly pre-existed from germinating seeds. Indeed,

335

glucosinolates are rich in broccoli seeds and their levels decrease during germination in

336

sprouts

337

dramatically reduced by 25 µM Na2SeO4 treatment (Figure 2B and Figure 2C). Particularly,

47

. In contrast, the total glucosinolate levels in young leaves and florets were


Page 15 of 26


338

the health beneficial glucosinolates glucoraphanin and neoglucobrassicin were greatly

339

suppressed (Figure 3B and Figure 3C). Other studies also report different plant organs

340

responding differently to Se treatments. While Se treatment leads to a decrease in

341

glucosinolate content in radish leaves, it causes a 35% increase in total glucosinolate content

342

in radish roots 35. Glucosinolate levels are known to be differently affected by Se based on

343

the dosages used. Se supplementation at low dosages exerts minimal effect on both total and

344

individual glucosinolate content in broccoli and other Brassica plants

345

application at higher dosages that even exert no negative effect on plant growth are reported

346

to reduce glucosinolate levels 16,23,34,39. Genotypic variation contributes to the variation in the

347

glucosinolate levels in response to Se treatments. Our previous study of 38 broccoli

348

accessions shows that approximately two third of them have similar total glucosinolate levels

349

when plants were exposed with or without 20 µM Na2SeO4 36. Kim and Juvik

350

different cultivars of broccoli with selenate and observed significant reduction in

351

glucoraphanin content in only two of the five genotypes. These studies indicate that broccoli

352

genotypes can be identified or bred in which glucosinolate biosynthesis (particularly

353

glucoraphanin) is not so sensitive to Se fertilization for producing broccoli with superior

354

chemopreventive properties.

29–31

. However, Se

31

treated five

355

Although the reduced total and individual glucosinolates by Se are often observed in

356

broccoli as well as in other Brassica plants, the basis underlying such a reduction is not fully

357

understood. Concomitantly, plant S nutrition is differently affected by Se based on the

358

dosages used 12–14. While Se at low dosages enhances S content, Se at elevated dosages exerts

359

an antagonistic relationship on plant S nutrition, which in turn could affect glucosinolate

360

metabolism

361

leaves and florets of broccoli was not due to reduced S levels. The S levels were comparable

362

in all three tissues of broccoli treated with and without 25 µM Na2SeO4 (Figure 4A). This

32–34

. We showed here that the Se-conferred glucosinolate reduction in young



Page 16 of 26

363

observation was consistent with a study showing that Se application causes no significant

364

change in total S content in two broccoli cultivars differing in glucosinolate content

365

Clearly, selenate treatment likely affected other metabolic processes, which influenced

366

glucosinolate metabolism.

29

.

367

A number of amino acids serve as precursors of glucosinolate biosynthesis 3.

368

Examination of these amino acid levels in broccoli tissues revealed that the levels of

369

methionine and phenylalanine were significantly reduced following selenate treatment in

370

young leaves and florets (Figure 5B-C). Methionine is the primary amino acid used for

371

aliphatic glucosinolate synthesis in Arabidopsis 2. It is possible that methionine also

372

contributes greatly to the aliphatic glucosinolate biosynthesis in broccoli tissues. Alteration of

373

methionine level via external supplementation has been shown to greatly enhance

374

glucosinolate levels in broccoli sprouts

375

might contribute to the negative effect of selenate treatment on glucosinolate content in

376

young leaves and florets of broccoli.

377

48

. The significant reduction of methionine level

Many genes in the glucosinolate biosynthetic pathway have been isolated and studied

378

3

379

glucosinolate biosynthesis in plants. MYB28 and MYB34 play key roles in regulating aliphatic

380

and indole glucosinolate biosynthesis, respectively

381

glucoraphanin content in Beneforte broccoli 51. Both MYB28 and MYB34 were significantly

382

down-expressed in young leaves and florets of two broccoli cultivars by selenate in our

383

studies (Figure 6 and Figure 7). The same associated expression of MYB28 with glucosinolate

384

content were also obtained in others studies

385

involved in methionine chair elongation for aliphatic glucosinolate synthesis. A BCAT4

386

knockout mutant gives approximately 50% reduction in aliphatic glucosinolates 52. Consistent

387

with reduced MYB28 expression, BCAT4 and MAM1 were also significantly downregulated

. In addition, some MYB transcription factors are identified which specifically regulate

49,50

. MYB28 is responsible for high

35

. BCAT4 and MAM1 are important genes


Page 17 of 26


388

in the selenate-treated leaves and florets. In addition, CYP79B2 and FMO2 expression was

389

significantly reduced in both young leaves and florets of these two broccoli cultivars. The

390

greatly suppressed expression of these two transcription factors along with some pathway

391

genes might be another factor that caused the inhibition of glucosinolate accumulation in the

392

Se-treated broccoli. Indeed, repression of genes involved in glucosinolate metabolism was

393

claimed to be the cause of Se conferred reduction of glucosinolate levels in radish leaves 35.

394

The effects of selenate on the global protein profile of broccoli florets were also

395

investigated through comparative proteomic analysis. The analysis revealed that selenate

396

treatment affected multiple cellular and metabolic processes. Particularly, proteins associated

397

with protein modification and metabolism were overrepresented following Se treatment. The

398

results corroborate other reports showing that proteins involved in these processes are among

399

the most enriched proteins in Se-treated tomato fruit

400

oxidative stresses and affects antioxidant enzyme activities

401

activities of ascorbate peroxidase, catalase and glutathione peroxidase was found to be

402

associated with Se suppressed plant growth

403

detected to be differentially expressed, consisting with no observed growth defect in the

404

selenate-treated broccoli at the dosage used.

53

. Se treatment is known to induce 13

. Reduced antioxidant enzyme

39

. None of these antioxidant proteins were

405

The alterations of various processes might indirectly affect glucosinolate metabolism

406

in the selenate-treated broccoli. The comparative proteomic analysis identified an enzyme

407

protein serine O-acetyltransferase participating in cysteine biosynthesis that was

408

down-regulated in the Se-treated broccoli. Cysteine serves as a precursor for methionine

409

synthesis. Methionine is the major precursor for aliphatic glucosinolate synthesis in

410

Arabidopsis 2. The suppressed level of this enzyme protein that affects cysteine and sulfur

411

metabolism might suggest a low capacity for methionine biosynthesis, which was consistent



412

with the observed reductions of methionine level and glucosinolate synthesis in the

413

Se-enriched broccoli florets.

414 415

AUTHORS’ CONTRIBUTION

416

Ming Tian, Fabricio W. Avila, and Tara Fish carried out most of the experiments. Yong Yang

417

and Theodore Thannhauser performed, analyzed, and/or supervised the proteomics work. Hui

418

Yuan did MapMan analysis. Maixia Hui, Siyi Pan, and Theodore Thannhauser helped with

419

data analysis. Ming Tian and Li Li designed the project and wrote the manuscript with

420

contributions from all the other authors.

421 422

ACKNOWLEDGEMENTS

423

We thank Shree Giri and Eric Craft for helping to analyze mineral concentrations by ICP.

424

Ming Tian and Maixia Hui acknowledge the support of the China Scholarship Council.

425 426

SUPPORTING INFORMATION

427

Figure S1. Levels of individual amino acid related to glucosinolate biosynthesis in florets

428

and leaves treated with and without Na2SeO4

429

Table S1. List of primer used in this study

430

Table S2. Differentially expressed proteins (>1.2 folds) between selenate treated and

431

untreated florets in three biological replicates

432

Table S3. Selenate up- and down-regulated proteins in MapMan functional groups

433

This material is available free of charge via the Internet at http://pubs.acs.org.

434 435 436 18 ACS Paragon Plus Environment

Page 18 of 26

Page 19 of 26


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594 595

Figure legends

596

Figure 1. Schematic view of the biosynthetic pathway of aliphatic and indolic glucosinolates

597

in plants. The core biosynthetic pathway genes are shown along the pathway. MYB28 and

598

MYB34 regulating the genes in the aliphatic and indole glucosinolate formation, respectively,

599

are indicated. Dotted arrows indicate multiple steps.

600 601

Figure 2. Total glucosinolate levels in sprouts (A), young leaves (B) and florets (C) of two

602

broccoli cultivars after treated with and without 25 µM Na2SeO4. Data represents means of

603

three biological replicates. Error bars indicate ± SD. Asterisks (*) show significant difference

604

between plants treated and non-treated with selenium (P ≤0.05). DW, dry weight

605 606

Figure 3. Individual glucosinolate levels in sprouts (A), young leaves (B) and florets (C) of

607

two broccoli cultivars treated with and without 25 µM Na2SeO4. Data represents means of

608

three biological replicates. Error bars indicate ± SD. Asterisks (*) show significant difference

609

between plants treated and non-treated with selenium (P ≤0.05). GR: Glucoraphanin; HGB:

610

Hydroxyglucobrassicin;

611

Neoglucobrassicin; GE: Glucoerucin

GB:

Glucobrassicin;

MGB:

Methoxyglucobrassicin;

NGB:

612 613

Figure 4. Total S (A) and Se (B) content in sprouts, young leaves and florets of two broccoli

614

cultivars treated with and without 25 µM Na2SeO4. Data represents means of four biological

615

replicates. Error bars indicate ± SD.

616 617

Figure 5. Amino acid content in sprouts, young leaves, and florets of two broccoli cultivars

618

treated with and without 25 µM Na2SeO4. (A) Total amino acids. (B) Methionine. (C) 24 ACS Paragon Plus Environment

Page 25 of 26


619

Tyrosine. (D) Phenylalanine. Data represents means of three biological replicates. Error bars

620

indicate ± SD. Asterisks (*) show significant difference between plants treated and

621

non-treated with selenium (P ≤0.05).

622 623

Figure 6. Expression of genes related to glucosinolate biosynthesis in young leaves of two

624

broccoli cultivars treated with and without 25 µM Na2SeO4. Data represents means of three

625

biological replicates. Error bars indicate ± SD. Asterisks (*) show significant difference

626

between plants treated and non-treated with selenium (P ≤0.05).

627 628

Figure 7. Expression of genes related to glucosinolate biosynthesis in florets of two broccoli

629

cultivars treated with and without 25 µM Na2SeO4. Data represents means of three biological

630

replicates. Error bars indicate ± SD. Asterisks (*) show significant difference between plants

631

treated and non-treated with selenium (P ≤0.05).

632 633

Figure 8. MapMan analysis displays the cellular processes affected by selenate in broccoli

634

florets. The numbers of up- and down-regulated proteins are

635


indicated.


636

TOC graphic

637

638


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Effects of selenium supplementation on glucosinolate biosynthesis in

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