Selenium biofortification in rice (Oryza sativa L.) sprouting: effects on

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Bioactive Constituents, Metabolites, and Functions

Selenium biofortification in rice (Oryza sativa L.) sprouting: effects on Se yield and nutritional traits with a focus on phenolic acid profile Roberto D\'Amato, Maria Chiara Fontanella, Beatrice Falcinelli, Gian Maria Beone, Elisabetta Bravi, Ombretta Marconi, Paolo Benincasa, and Daniela Businelli J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00127 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 6, 2018

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Journal of Agricultural and Food Chemistry

Selenium biofortification in rice (Oryza sativa L.) sprouting: effects on Se yield and nutritional traits with a focus on phenolic acid profile

Roberto D’Amatoa, MariaChiara Fontanellab, Beatrice Falcinellia, GianMaria Beoneb, Elisabetta Bravia, Ombretta Marconia, Paolo Benincasa*,a, and Daniela Businellia

a

Department of Agricultural, Environmental and Food Science, University of Perugia, Perugia,

Italy b

Department for Sustainable Process, Agricultural Faculty, Università Cattolica del Sacro Cuore of

Piacenza, Piacenza, Italy. *

Corresponding author: Paolo Benincasa tel +39 0755856325

1 ACS Paragon Plus Environment


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1

ABSTRACT

2

The contents of total Se and of inorganic and organic Se species, as well as the contents of proteins,

3

chlorophylls, carotenoids and phenolic acids were measured in 10-day old sprouts of rice (Oryza

4

sativa L.) obtained with increasing levels (15, 45, 135, and 405 mg Se L-1) of sodium selenite and

5

sodium selenate and with distilled water as control. Increasing Se levels increased organic and

6

inorganic Se contents of sprouts, as well as the content of phenolic acids, especially in their soluble

7

conjugated forms. Moderate levels of sodium selenite (i.e., not higher that 45 mg L-1) appeared the

8

best compromise to obtain high Se and phenolic acid yields together with high proportion of

9

organic Se, while limiting residual Se in the germination substrate waste. Se biofortification of rice

10

sprouts appears a feasible and efficient way to promote Se and phenolic acid intake in human diet,

11

with well known health benefits.

12 13

KEYWORDS: sprout, selenium species, phenolic acid, carotenoid, chlorophyll

14


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15

INTRODUCTION

16

Selenium (Se) is an essential micronutrient for mammals, involved in major metabolic pathways,

17

like the production of a wide range of selenoproteins (i.e., selenocysteine, selenomethionine, etc.),

18

the thyroid hormone metabolism and the immune functions.1 The low Se status is associated with

19

several health diseases (i.e., Keshan disease, heart diseases, hyperthyroidism, enhanced

20

susceptibility to infections, cancer),1,2 and occurs in geographic areas where the soil is particularly

21

poor in this element and, consequently, the food is poor as well. The Se supply in almost all

22

European countries is below the recommended daily intake (50-55 µg Se).3

23

The procedure aimed at increasing the selenium content in plants during early stages is known as

24

Se-biofortification and has been developed for several plants including trees4 and vegetables.5

25

Within the existing compounds suitable for Se-biofortification, inorganic ones (e.g., sodium selenite

26

and sodium selenate) are known to be cheap and efficient, whereas organic ones (i.e.,

27

selenoproteins) are expensive although more relevant for human nutrition. However, plants are able

28

to produce selenoproteins starting from inorganic Se compounds,1 thus inorganic forms might be

29

preferred because cost-effective.

30

Se-biofortification has been recently applied for the production of Se-enriched sprouts.6–9 Sprouts,

31

the young seedlings obtained a few days after seed germination, are a high value food produced

32

homemade or for the ready-to-eat market.10 Compared to seeds, sprouts have higher nutritional

33

traits, such as an increased content of phytochemicals, i.e., the secondary metabolites of plants,

34

known to provide several benefits for human health.10 Seeds during germination are able to absorb

35

high amounts of Se.8,11

36

Rice (Oryza sativa L.) is a basic food all over the world and gluten free,12 which makes it

37

particularly appreciated in light of the increasing incidence of coeliac disease. Some literature exists

38

on Se-biofortification in rice sprouts, but this was limited to low Se concentrations (from 10 to 40

39

mg Se L-1) of sodium selenite.6,11,12 No literature considered the effect of sodium selenite at

40

concentrations over 40 mg Se L-1 and the effect of sodium selenate. Moreover, only Chomchan et 3 ACS Paragon Plus Environment


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41

al.12 investigated the effect of Se-biofortification on sprout phenolic content, and they measured

42

only the total phenolic content, with no focus on specific compounds, like phenolic acids. Phenolic

43

acids (PAs) are well represented in cereal species13 and have been found in rice sprouts, with

44

different chemical forms,14 implicating different bioavailability and health benefits. Soluble free

45

(i.e., aglycones) and conjugated (i.e., ester- and glycoside-bound) PAs are easily available for

46

absorption in the human large and small intestines, whereas bound ones (i.e., cell wall-bound)

47

overcome digestion in the stomach and small intestine. For this reason, bound PAs help reduce the

48

risk of colorectal cancer and provide site-specific health benefits in the colon and other tissues after

49

absorption.15 Several pigments are also reported to have health promoting effects. Among these,

50

carotenoids,16 chlorophyll and its derivatives.17

51

Since recent research demonstrated that increasing concentrations of sodium chloride increase PAs

52

content in cereals18 as well as in other species,19 similarly, sodium selenite and selenate might be

53

assumed to have an effect on PAs content of sprouts. A wider picture on the effect of these salts

54

might include the measurement of possible variations in the content of pigments.

55

Thus, in this research we germinated rice grains and grew seedlings in hydroponics at increasing

56

concentrations of sodium selenite or sodium selenite to evaluate: i) sprout growth; ii) Se

57

accumulation in sprouts as organic and inorganic forms; iii) Se optimal concentrations and

58

biofortification efficiency; iv) Se biofortification effects on sprouts nutritional traits with a focus on

59

soluble free, soluble conjugated and insoluble bound phenolic acids, and on some pigments.

60 61

MATERIALS AND METHODS

62

Chemicals

63

Sodium selenite (Na2SeO3), sodium selenate (Na2SeO4), selenomethionine (SeMet), selenocystine

64

(SeCys2), Se-(Methyl)selenocysteine (SeMeSeCys), protease (Protease Type XIV), purchased from

65

Sigma (St. Louis, MO). Ammonium acetate (RPE for analysis), nitric acid (HNO3 65% RPE)

66

hydrogen peroxide (H2O2 40% wv RE pure), salicylic acid, methanol (HPLC grade), acetonitrile 4 ACS Paragon Plus Environment

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(HPLC grade), water (HPLC grade), orthophoshoric acid 85% (RPE grade), sodium laurylsulphate

68

(SLS, RS grade), and potassium monobasic phosphate (RP-ACS grade) were purchased from Carlo

69

Erba (Milan, Italy). HPLC-grade water was obtained from ultrapure water purification system (18.2

70

MΩ cm, ELGA PURELAB flex, Veolia Water Solutions and Technologies, Ontario, Canada). ICP-

71

MS germanium solution (72Ge, Li6, 45Sc, 89Y, 159 Tb, 209 Bi) was purchased from Agilent

72

(Santa Clara, California, USA). Gallic acid (GA), α-resorcylic acid (3,5-dihydroxybenzoic acid) (α-

73

RA), gentisic acid (GeA), vanillic acid (VA), salicylic acid (SaA), p-coumaric acid (p-CA), ferulic

74

acid (FA), and o-coumaric acid (o-CA) were purchased from Fluka (Buchs SG, Switzerland).

75

Sinapic acid (SiA) was purchased from Carl Roth GmbH and Co. (Karlsruhe, Germany).

76 77

Plant material and experimental design

78

Rice (Oryza sativa L., cv. Selenio) grains were provided by Ente Nazionale Risi (Milano, Italy).

79

Grains (15 g) were sown in plastic trays with distilled water (Se_0) and sodium selenite (SeIV) or

80

sodium selenate (SeVI) solutions at 15, 45, 135 and 405 mg Se L-1. Trays were incubated in a

81

growth chamber at 20-28 °C in a dark/light regime of 10:14 h and light intensity of 200 µmol m-2 s-

82

1 20

83

each replicate were harvested 10 days after sowing (DAS) and roots were washed with distilled

84

water to remove the selenium sticking to, but not absorbed by, the roots. Roots were then wiped

85

with blotting paper and separated from shoots. No sprout was obtained from SeVI_405 since no

86

seed was able to germinate. Fresh and dry mass (FM and DM respectively) of sprouts and shoot and

87

root lengths from each treatment were measured on ten individuals per replicate. The dry mass was

88

determined after 24 hours at 60 °C for 24 h in a ventilated stove. The lengths were measured using a

89

micrometer having an accuracy of 0.02 mm.

90

A sample of the total fresh shoots from each replicate was immediately used for Se speciation and

91

for the determination of chlorophylls, carotenoids and proteins. The remaining shoot sample, as

92

well as the total root sample, were dried at 45 °C for 48 h, finely powdered, and stored at room

,

according to a completely randomized design with three replicates (trays). Rice sprouts from



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93

temperature for the determination of total Se concentration. Three separate subsamples were taken

94

from each replicate, and each subsample was analysed once. Therefore the mean value of each

95

treatment is the result of nine separate measurements (i.e., 3 replicates per treatment x 3 subsamples

96

per replicate x 1 measurement per subsample).

97 98

Total selenium content

99

Total Se content (TSeC) was determined following the method of Cubadda et al.21 Dried shoots and

100

roots (0.25 g) were microwave digested (ETHOS One high-performance microwave digestion

101

system; Milestone Inc., Sorisole, Bergamo, Italy) with 8 mL of ultrapure concentrated nitric acid

102

(65% w/w) and 2 mL of hydrogen peroxide (30% w/w). The heating program for the digestion

103

procedure was 30 min with power of 1000 W and 200 °C. After cooling down, the digests were

104

diluted with distilled water up to 20 mL and analysed by ICP-MS (Agilent 7900, Agilent

105

Technologies, USA) with Octopole Reaction System (ORS) (Table S1, Supporting Information).

106

Total Se standard solutions were prepared by diluting the corresponding stock solutions (Selenium

107

standard 1000 mg L-1 for AAS TraceCert Sigma Aldrich 89498, Milan, Italy) with HPLC-grade

108

water. Results were expressed as micrograms per grams of dry mass (µg g-1 DM). The ratio between

109

TSeC in shoots and roots was also determined.

110 111

Se speciation

112

Fresh shoot material (0.25 g) was mixed with 10 mL of solution 2.0 mg mL-1 of protease. Samples

113

were sonicated with an ultrasound probe for 2 min, and stirred in water bath at 37 °C for 4 h. Then,

114

samples were cooled at room temperature and centrifuged for 10 min at 9000 rpm. The supernatant

115

was filtered through 0.22 µm Millex GV filters (Millipore Corporation, Billerica, MA). The

116

standards solutions (1, 5, 10 and 20 µg L-1) for inorganic (i.e., selenite, SeO3-2 and selenate, SeO4-2)

117

and

118

selenomethionine, SeMet. Se forms were prepared with ultrapure (18.2 MΩ cm) water.

organic

(i.e.,

selenocystine,

SeCys2;

Se-(Methyl)selenocysteine,

(SeMeSeCys);


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119

Speciation of Se was performed by HPLC (Agilent 1100, Agilent Technologies, USA) using an

120

anion exchange column (Hamilton, PRP-X100, 250 x 4.6 mm, 5 µm particle size). The mobile

121

phase was made by ammonium acetate with gradient elution. The analytical method and

122

instrumental conditions were previously described in Fontanella et al.22 Chromatographic

123

conditions and performance parameters of ICP-MS are reported in Table S1 and S2 (Supporting

124

Information), respectively. Results were expressed as micrograms per grams of dry mass (µg g-1

125

DM). Total inorganic and organic Se content were calculated as the sum of single inorganic (i.e.,

126

SeO3-2 and SeO4-2) and organic (i.e., SeCys2, SeMeSeCys and SeMet) compounds, respectively.

127 128

Protein content

129

Protein concentration on fresh shoot sample was determined using Bradford assay.23 The fresh

130

shoot material (0.5 g) was crushed in a mortar using 100 mL of Tris-HCl 0.05 M, pH 9.0. The

131

extract was centrifuged at 9000 rpm and the supernatant (100 µL) was mixed with Bradford reagent

132

(5000 µL) for 1 min using a vortex. After 10 min the absorbance was measured at 595 nm. Bovine

133

serum albumin (BSA) was used as standard and results were expressed as mg of BSA per gram of

134

dry mass (mg BSA g-1 DM).

135 136

Total chlorophyll and carotenoid contents

137

The contents of chlorophylls (Chlorophyll a, Ca and Chlorophyll b, Cb) and total carotenoids (TCC)

138

were determined following the method of Lichtenthaler and Wellburn.24 Two grams of fresh shoots

139

were dissolved in 25 mL of 80% acetone in water. The solution was filtered through a double layer

140

of cheese cloths and the absorbance was read at 663.2, 646.5, and 470 nm using a UV/VIS

141

spectrometer Lamda EZ 150 Perkin Elmer (Waltham, MA, USA), having a resolution of 0.1 nm.

142

Chlorophylls and carotenoids content were calculated as follows:

143

Ca (mg L-1) = (12.25 A663,2) – 2.79 A646,5

144

Cb (mg L-1) = (21.5 A646,5) – 5.1 A663,2 7 ACS Paragon Plus Environment


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TCC (mg L-1) = (1000 A470 – 18.2 Ca – 85.02 Cb)/198

145 146

The sum of Ca and Cb was reported as the total content of chlorophylls (TChlC) and the ratio

147

between TChlC and TCC (TChlC/TCC) was also calculated. Results were expressed as milligrams

148

per gram of dry mass (mg g-1 DM).

149 150

Phenolic acids contents

151

The phenolic acids contents were measured using a modified method by Krygier et al.25 and Adom

152

and Liu15 as follows. About 1 g of fresh shoot was extracted with 10 mL methanol/acetone/water

153

(7:7:6, v/v/v) and divided in three fractions: soluble free, soluble conjugated, and bound phenolic

154

acids. For each fraction, nine phenolic acids were determined: gallic (GA), α-resorcilic (α-RA),

155

gentisic (GeA), vanillic (VA), salicylic (SaA), p-coumaric (p-CA), ferulic (FA), sinapic (SiA), and

156

o-coumaric (o-CA).

157

The chromatographic separation was achieved at room temperature with an Eurospher II C18

158

reverse-phase column (Knauer 150 mm x 4 mm ID, Knauer, Berlin, Germany). The three

159

wavelengths for the determination of phenolic acids were 254, 278, 324 nm. All procedures were

160

carried out, adapting the method of Floridi et al.26 Mobile phase A was 0.05 M KH2PO4 and 0.05

161

µM sodio lauryl sulphate (SLS), and mobile phase B was phase A/CH3OH/CH3CN, 30:20:50 v/v/v,

162

0.05 µM SLS. The mobile phases were adjusted to pH 3.25 with 85% orthophosphoric acid and

163

were filtered with a 0.22 µm membrane filter (Millipore, Bedford, MA, for aqueous solvents; MSI,

164

MA, for organic solvents). The solvent gradient and flow rate during analysis are reported in Table

165

S3 (Supporting Information).

166

The following equipment was utilized for the HPLC analysis: a quaternary Azura P 6.1 L pump

167

(Knauer, Berlin, Germany), a Knauer 3950 autosampler with a 10-µL loop, and an Azura MWD 2.1

168

L (Knauer, Berlin, Germany) DAD detector. The system was managed by Clarity Chromatography

169

Software for Windows (DataApex, Prague, Czech Republic).

170 8 ACS Paragon Plus Environment

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Statistical analysis

172

Data were subjected to the analysis of variance according to a randomized blocks desing with three

173

replicates. Average values of triplicate determinations for n = 3 independent replicates ± standard

174

deviation are depicted. Means were compared by using the Fisher’s Least Significant Difference

175

(LSD) at P = 0.05.

176 177

RESULTS

178

Growth indexes

179

The individual sprout growth was affected by Se form and concentration in the substrate (Table 1).

180

Compared to Se_0, the FM increased in SeIV_15 (+52%) and in SeVI_15 (+15%), while it

181

decreased at higher Se concentrations, slightly for SeIV (from -10% in SeIV_45 to -44% in

182

SeIV_405), markedly for SeVI (on average about -84%). The % dry matter content of sprouts did

183

not substantially change among treatments, except for a slight increase in SeVI_135, thus the trend

184

of DM was similar to that of FM. As described in Materials and Methods, no sprout was obtained

185

from SeVI_405 because germination was completely inhibited by this treatment.

186

Shoot length was not affected by SeIV except for a moderate decrease in SeIV_405 (-21%

187

compared to S_0), while it was always decreased by SeVI (from -12% in SeVI_15 to -38% in

188

SeVI_135, compared Se_0). Root length showed the same trend of FM, i.e., it increased in SeIV_15

189

and SeVI_15, while it decreased at higher Se concentrations especially in SeVI.

190 191

Selenium and protein contents

192

Total selenium content (TSeC) increased in all Se treatments compared to Se_0 (Table 2). In shoots,

193

TSeC increased at increasing SeIV concentrations up to 64-fold higher than Se_0 in SeIV_405,

194

while it reached a maximum in SeVI_45 (119-fold than Se_0) and then decreased. In roots, TSeC

195

increased at increasing Se concentrations both for SeIV (up to 62-fold than Se_0 in SeIV_405) and



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196

SeVI (up to 12-fold than Se_0 in SeVI_135). The shoot/root ratio for TSeC was slightly affected by

197

SeIV, whereas it was markedly increased by SeVI treatments.

198

As far as the contents of inorganic Se species in shoots are concerned, the SeO32- content increased

199

with increasing level of SeIV and, especially, SeVI (Table 3). The SeO42- was not detected in shoots

200

of SeIV treatments except for very low concentrations at the highest levels, while it was very high

201

in shoots of SeVI treatments, with a maximum for those of SeVI_45 (Fig. 1). Thus, for a given Se

202

level, the total inorganic Se content was lower with SeIV than with SeVI.

203

Organic Se forms were non detectable in Se_0, while they increased with increasing Se level in

204

both SeIV and SeVI treatments (except for SeCys2 in SeVI, which was maximum for SeVI_45)

205

(Table 3). For a given Se level, SeCys2 and SeMet, as well as total organic Se forms, were generally

206

lower in SeIV than in SeVI treatments, although SeMeSeCys was lower in SeVI.

207

Protein content was slightly affected by SeIV treatments, whereas it decreased with increasing SeVI

208

treatments (more than halved in SeVI_135 compared to Se_0).

209 210

Total chlorophyll and carotenoid contents

211

Total chlorophyll (TChlC) was increased by low SeIV levels (i.e., +20% on average of SeIV_15

212

and SeIV_45, compared to Se_0) and decreased by higher SeIV levels and by all SeVI levels (Table

213

4). Total carotenoid content (TCC) did not change among Se_0, SeIV_15 and SeVI_15, whereas it

214

was decreased by higher concentrations of both. The TChlC/TCC ratio was generally higher in

215

SeIV than in SeVI and reached a maximum for SeIV_45 (+45% compared to Se_0).

216 217

Phenolic acid contents

218

Soluble free and soluble conjugated PAs were generally increased by SeIV and SeVI treatments as

219

compared to Se_0, while bound PAs showed irregular variations (Table 5). In most cases, soluble

220

free PAs showed moderate variations (except for the high increases in SeIV_135 and, especially,


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SeIV_405), as well as bound PAs, while soluble conjugated PAs showed dramatic increases

222

compared to Se_0.

223

As far as single PAs are concerned, Se_0 showed only the three forms of GA, plus soluble

224

conjugated SaA, and bound α-RA, GeA, FA. Both SeIV and SeVI caused the appearance of soluble

225

free SaA, soluble conjugated GeA, VA, p-CA, FA, SiA, and bound SaA, but the presence and the

226

content of these PAs varied with the Se level, without a clear trend. Moreover, free FA appeared at

227

SeIV_405 and o-CA at SeIV_135 and SeIV_405. Soluble conjugated SaA reached very high

228

concentrations in most SeIV and SeVI treatments, and soluble conjugated FA reached very high

229

concentrations in SeVI treatments.

230 231

DISCUSSION

232

Results on sprout growth parameters suggest some consideration about the effect of the forms and

233

levels of selenium (Table 1). The increase of sprout mass and root length for selenite and selenate at

234

the lowest Se level (i.e., 15 mg Se L-1) would suggest a stimulatory effect, a sort of a hormetic

235

effect.27 The decrease of all growth parameters at higher Se levels could be due to a toxic effect of

236

Se on rice sprouts.28 A reduction of growth for Se (as selenite) levels of 25 mg L-1 or higher was

237

also observed for chickpea sprouts by Zhang et al.29 The toxic effect of Se would be greater in case

238

of selenate, which caused a delay of germination and growth in SeVI_135 and the complete lack of

239

germination in SeVI_405. The slightly higher dry matter % content of sprouts in SeVI_135 was due

240

to their earlier growth stage, so that the proportion of the grain compared to roots and shoots was

241

still important (Table 1). The higher phyto-toxicity of selenate compared to selenite was also

242

observed in 19 day-old rice plants by Nothstein et al.30, who hypothesized that, in a growing

243

substrate without nutrients, toxicity might be linked to the competition between Se and essential

244

ions for transporter proteins.

245

The increase of TSeC with increasing Se levels (Table 2) is in line with results of Chomchan et al.12

246

for rice shoots 8-day old and of Nothstein et al.30 for rice plantlets 19-day old. The decrease of 11 ACS Paragon Plus Environment


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TSeC in shoots passing from SeVI_45 to SeVI_135 is a consequence of the above said delayed seed

248

germination and seedling growth (Table 1), which caused a lower Se absorption. The literature on

249

sprout biofortification with selenate is limited to very low Se levels (i.e., up to 15 mg L-1),30,31 while

250

no reference is available for high Se levels, such as those used here.

251

Results on TSeC ratio between shoots and roots (Table 2) are in line with existing literature on

252

rice30 and other cereal species.32,33 These results confirm that selenite application causes Se

253

accumulation mainly in roots, while selenate causes Se accumulation mainly in shoots. This

254

because with selenite application Se is taken up by roots through passive diffusion34 and it is

255

accumulated or transformed there into organic Se.30,32,33 On the contrary, selenate causes Se uptake

256

by roots through the sulphate transporters, due to its chemical similarities with sulphur1, and then Se

257

is easily transferred to shoots via the xylem.30,32,33

258

The contents of Se species were measured only in shoots (Table 3), as they represent the edible

259

portion of 1 to 2-week old sprouts for most cereal species.20 The lack of SeO4-2 in shoots in case of

260

selenite application was expected and is in line with findings by Nothstein et al.30 In fact, it has been

261

hypothesized that selenite is transported to shoots as SeO3-2 via the xylem or as organic Se

262

compounds (i.e., SeMet, SeMeSeCys, etc.) via the phloem.30,35 In any case, the plant would not

263

need the SeO42- form. Conversely, in plants grown with selenate, the SeO4-2 taken up by roots is

264

rapidly transported from roots to shoots (i.e., in the chloroplasts)29 where it is reduced to SeO3-2 and

265

used to produce seleno-proteins.1 It is of relevance that for a given Se level, the use of selenate

266

allowed contents of SeO3-2 and organic Se species in shoots similar (at high Se treatments) or even

267

higher (at low Se treatments) compared to selenite, and, in addition, a much higher SeO4-2

268

accumulation. From the nutritional point of view, organic Se (e.g., SeMet) is absorbed and retained

269

more efficiently in humans than inorganic forms, thus organic Se (especially SeMet, the major

270

species in food) is more recommendable than inorganic Se in the frame of a balanced diet.36,37 With

271

this regard, the treatments with selenite up to SeIV_135 gave a much lower inorganic to organic Se

272

ratio than treatments with selenate, thus selenite should be preferred in the production of Se 12 ACS Paragon Plus Environment

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biofortified sprouts. In particular, SeIV_15 and especially SeIV_45 gave a good Se yield (i.e., the

274

product between Se concentration in shoots and their harvested biomass) in front of a low amount

275

of selenite applied to the substrate. Higher levels of selenite treatments (e.g., SeIV_135) appear not

276

advisable for several reasons: i) they may delay germination and depress growth, which complicates

277

the sprouting process; ii) the higher the Se level of the treatment the higher the residual Se in the

278

germination substrate waste, which is not convenient economically; iii) given the total Se

279

concentration in sprouts of high selenite treatments (Table 3) and a dry matter concentration of

280

around 15% (Table 1), a consumption of just 100 g of fresh sprouts would exceed the tolerable

281

upper level of daily Se intake in humans (0.4 mg),38 while a precautionary lower level is

282

recommended.

283

The most represented species of organic Se (Table 3) in our experiment were SeMet and

284

SeMeSeCys, i.e., the two species observed in previous experiments in rice grown with Se.11,35 Both

285

of them are produced from selenocysteine (SeCys), the first compound obtained in Se assimilation.1

286

When the plant tissue is broken for the preparation of the extract, the oxygen of the air causes

287

SeCys conversion to its dimer SeCys2, which is the detected compound. The high levels of SeCys2

288

and SeMet observed at high Se levels could implicate selenium toxicity,39 because their

289

incorporation into proteins instead of cysteine and methionine, respectively (i.e. the correspondent

290

Sulphur compounds) may cause metabolic dysfunction.1 The high content of SeMeSeCys,

291

nonprotein selenoaminoacid, at high Se levels would represent a strategy to avoid the incorporation

292

of Se into proteins and thus to avoid Se toxicity.1 In fact, high levels of SeMeSeCys in plant tissues

293

occur mainly in Se-accumulator plants,40 and have been observed in rice grown with selenite.11

294

The decrease of proteins with increasing selenate levels may be explained with the interference of

295

Se on sulphur assimilation and thus on aminoacid synthesis.1

296

As far as pigments are concerned, the decrease of both chlorophyll and carotenoid contents (Table

297

4) at high Se levels may be due to the down regulation effect of Se on porphobilinogen synthetase,41

298

required for chlorophyll biosynthesis, and on phytoene synthase (PSY), the key enzyme for 13 ACS Paragon Plus Environment


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299

carotenoid production.42 On the other hand, the slight increase of chlorophyll content observed at

300

low Se levels might arise from the positive effect of Se in chlorophyll biosynthesis through the

301

promotion of electron flux in respiration43,44 and the protection of chloroplast enzymes.41,45

302

Chomchan et al.12 did not observe substantial effect of selenite on pigment contents.

303

With regards to phenolic acids (Table 5), the presence of GA, SaA and FA, in rice sprouts and

304

ricegrass was reported by many authors,12,14,46 but there is no evidence yet for the effect of Se on

305

these and other PAs like α-RA, GeA, p-CA, SiA and o-CA. Moreover, no literature is available in

306

rice sprouts for different PAs forms except for Ti et al.,14 who considered free and bound PAs but

307

not the soluble conjugated ones.

308

The results we obtained for the different forms of PAs at increasing Se levels may be explained

309

taking into account the effect of Se on the production of reactive oxygen species (ROS) and

310

activation of L-phenylalanine ammonia lyase (PAL). Wang et al.47 demonstrated that selenite

311

increased ROS production in peanut seedlings, while Guardado-Félix et al.48 demonstrated that

312

selenite increased PAL activity and some phenolics like isoflavones in chickpea. Thus the presence

313

of selenium would represent an abiotic stress similar to that caused by other heavy metals and the

314

plant would face it by activating the phenylpropanoid pathway27 for producing phenolic

315

compounds, including PAs,49 which may improve ROS scavenging. This could explain both the

316

appearance of new PAs and the increase of some PAs already present, like FA and SaA. However,

317

literature about the effect of Se on phenolic content is not always unanimous,1 with increases or

318

decreases of total polyphenols and PAs, which could arise from differences in plant species, plant

319

growth stage and selenium treatments. An increase of free and conjugated forms of PAs and a

320

concurrent decrease of bound PAs were observed in rapeseed sprouted under NaCl stress by

321

Falcinelli et al.19 The increase of soluble free and soluble conjugated PAs is of relevance because

322

these forms may be more rapidly absorbed during digestion and released in the body, inhibiting

323

oxidation of low-density lipoprotein (LDL) cholesterol and liposomes.50


Page 15 of 31


324

Among PAs, SaA and FA showed the highest increase in the presence of Se. SaA is well known to

325

play a role in improving plant tolerance to major abiotic stresses like those produced by heavy

326

metals, salinity, water deficiency, and high temperature,51 The benefits for human health associated

327

to SaA are well known,52 as well as those of FA,53 VA,54 GeA55 and p-CA.20

328

In conclusion, Se-biofortification with increasing Se levels increased both organic and inorganic Se

329

content of sprouts, as well as the content of phenolic acids, especially in their soluble conjugated

330

forms. Moderate levels of sodium selenite (i.e., not higher that 45 mg L-1) appear the best

331

compromise to obtain high Se and phenolic acid yields (i.e. the product between biomass and Se or

332

phenolic acid concentrations) together with high proportion of organic selenium (the most effective

333

in human nutrition), while limiting residual Se in the germination substrate waste. Se-

334

biofortification of rice sprouts comes out as a feasible and efficient way to increase the potential Se

335

intake in human diet and achieve the health benefits associated to both Se and phenolic acids.

336 337

ABBREVIATIONS USED:

338

Se, selenium; PAs, phenolic acids; SeMet, selenomethionine; SeCys2, selenocystine; SeMeSeCys,

339

Se-(Methyl)selenocysteine; RPE, reagent analytical grade; RE, reagent technical grade; RS, special

340

reagents; GA, gallic acid; α-RA, α-resorcylic acid; GeA, gentisic acid; VA, vanillic acid, SaA,

341

salicylic acid, p-CA, p-coumaric acid; FA, ferulic acid; o-CA, o-coumaric acid; SiA, sinapic acid;

342

SeIV, sodium selenite; SeVI, sodium selenate; DAS, days after sowing; FM, fresh mass; DM, dry

343

mass; TSeC, total selenium content; SeO3-2, selenite; SeO4-2, selenate; BSA, bovine serum albumin;

344

Ca, chlorophyll a; Cb, chlorophyll b; TCC, total carotenoid content; TChlC, total chlorophyll

345

content; SLS, sodium lauryl sulphate; PSY, phytoene synthase; ROS, reactive oxygen species;

346

PAL, phenylalanine ammonia lyase; LDL, low-density lipoprotein

347 348

ACKNOWLEDGEMENTS

349

We gratefully acknowledge Mr Silvano Locchi for his help in the sprouting of rice in the seed lab. 15 ACS Paragon Plus Environment


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350 351

SUPPORTING INFORMATION

352

Table S1. Instrumental conditions for ICP-MS and HPLC system.

353

Table S2. Performance parameters of ICP-MS.

354

Table S3. Solvent gradient and flow rate for phenolic acids HPLC determination.

355 356

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FIGURE CAPTIONS

509 510

Figure 1. HPLC-ICP-MS chromatogram of samples from rice sprouts fortified with sodium selenite

511

(SeIV_45, solid line) and sodium selenate (SeVI_45, dotted line). Peak identities: A = SeCys2; B=

512

SeMetSeCys, C= SeMet, D=SeO32-, E= SeO42-.

513



Page 24 of 31

TABLES

Table 1. Individual Total Fresh Mass and % Dry Matter Concentration and Shoot and Root Lengths of 10-Day Old Rice Sprouts Obtained with Distilled Water (Se_0) or at 15, 45, 135 and 405 mg L-1 of Sodium Selenite (SeIV_15, SeIV_45, SeIV_135 and SeIV_405) and 15, 45, 135 mg L-1 of Sodium Selenate (SeVI_15, SeVI_45, SeVI_135). Average Values for n=3 Independent Replicates are reported. Standard Deviations in Brackets. Different Letters within each Column Indicate Statistically Significant Differences at P < 0.05.

Fresh Mass (g)

Dry matter concentration (%)

Shoots length (mm)

Root length (mm)

Se_0

0.65 (0.005) c

15.47 (0.21) b

43 (1.5) b

66 (1.7) c

SeIV_15 SeIV_45 SeIV_135 SeIV_405

0.99 (0.025) a 0.58 (0.023) d 0.44 (0.039) e 0.36 (0.026) f

14.73 (0.32) d 14.77 (0.15) cd 15.17 (0.15) bc 14.60 (0.26) d

44 (1.2) ab 46 (4.4) ab 43 (0.6) ab 34 (2.1) d

99 (1.7) a 59 (4.0) d 46 (3.0) e 36 (2.5) f

SeVI_15 SeVI_45 SeVI_135

0.75 (0.058) b 0.12 (0.007) g 0.09 (0.004) g

13.93 (0.15) e 14.07 (0.25) e 17.60 (0.46) a

38 (1.2) c 31 (1.5) d 26 (1.5) e

74 (4.4) b 11 (2.5) g 7 (1.0) g

Treatment


Page 25 of 31


Table 2. Total Selenium Content (TSeC) in Shoots and Roots and Shoot/Root Ratio of TSeC in 10Day Old Rice Sprouts Obtained with Distilled Water (Se_0) or at 15, 45, 135 and 405 mg L-1 of Sodium Selenite (SeIV_15, SeIV_45, SeIV_135 and SeIV_405) and 15, 45, 135 mg L-1 of Sodium Selenate (SeVI_15, SeVI_45, SeVI_135). Average Values for n=3 Independent Replicates, each Analysed in Triplicate, are reported. Standard Deviations in Brackets. Different Letters within each Column Indicate Statistically Significant Differences at P < 0.05.

Treatments

TSeC (µg g-1DM) Shoots Roots

TSeC shoot/ root ratio

Se_0

0.76 (0.09) g

1.37 (0.16) h

0.55 (0.04) e


18.78 (0.39) f 23.61 (0.35) e 36.39 (1.00) d 48.67 (0.51) c

28.13 (1.47) d 36.15 (2.05) c 65.77 (1.60) b 85.17 (1.59) a

0.67 (0.05) d 0.65 (0.03) d 0.55 (0.00) e 0.57 (0.01) e


18.40 (1.00) f 90.51 (2.12) a 66.50 (1.17) b

3.76 (0.49) g 14.65 (2.08) f 17.10 (0.50) e

4.96 (0.80) b 6.25 (0.83) a 3.89 (0.18) c



Page 26 of 31

Table 3. Inorganic and Organic Se Species and Protein Content in Shoots of 10-Day Old Rice Sprouts Obtained With Distilled Water (Se_0) or at 15, 45, 135 and 405 mg L-1 of Sodium Selenite (SeIV_15, SeIV_45, SeIV_135 and SeIV_405) and 15, 45, 135 mg L-1 of Sodium Selenate (SeVI_15, SeVI_45, SeVI_135). Average Values for n=3 Independent Replicates, each Analysed in Triplicate, are reported. Standard Deviations in Brackets. Different Letters within each Column Indicate Statistically Significant Differences at P < 0.05.

Treatments

Proteins (mg BSA g-1 DM)

Se species (µg g-1 DM)

SeO3

2-

Inorganic species SeO42-

Total

SeCys2

Organic species SeMet SeMeSeCys

Total

Se_0

0.02 (0.01) g

< LOD

0.02 (0.01) h

< LOD

< LOD

0.06 (0.01) h

0.06 (0.01) f

110 (1.75) b


0.98 (0.03) f 1.58 (0.16) e 3.62 (0.28) d 8.49 (0.17) a

< LOD < LOD 0.08 (0.01) d 0.06 (0.02) de

0.98 (0.03) g 1.58 (0.16) f 3.70 (0.27) e 8.55 (0.18) c

1.24 (0.20) e 1.65 (0.08) d 2.03 (0.08) c 2.63 (0.19) b

2.55 (0.13) f 10.1 (0.28) c 13.2 (0.9) a 11.2 (0.59) b

2.80 (0.23) g 5.62 (0.42) f 8.62 (0.44) d 10.3 (0.45) b

6.58 (0.50) e 17.4 (0.75) c 23.8 (0.59) b 24.1 (0.05) ab

107 (1.65) bc 130 (2.82) a 108 (0.49) bc 103 (2.16) c


1.66 (0.20) e 5.64 (0.24) c 7.59 (0.29) b

3.95 (0.11) c 57.2 (1.34) a 44.1 (1.54) b

5.61 (0.31) d 62.8 (1.10) a 51.7 (1.39) b

1.34 (0.09) e 3.55 (0.10) a 2.48 (0.12) b

1.57 (0.07) g 4.86 (0.09) e 7.81 (0.13) d

6.46 (0.37) e 9.51 (0.24) c 14.6 (0.12) a

9.37 (0.45) d 17.9 (0.35) c 24.9 (0.18) a

92.3 (1.37) d 78.5 (1.85) e 51.9 (3.32) f

SeO32-: selenite; SeO42-: selenate; SeCys2: selenocystine; SeMeSeCys: Se-(Methyl)selenocysteine; SeMet: selenomethionine;

26

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Table 4. Total Chlorophyll (TChlC) and Carotenoid (TCC) Contents and TChlC/TCC Ratio in Shoots of 10-Day Old Rice Sprouts Obtained with Distilled Water (Se_0) or at 15, 45, 135 and 405 mg L-1 of Sodium Selenite (SeIV_15, SeIV_45, SeIV_135 and SeIV_405) and 15, 45, 135 mg L-1 of Sodium Selenate (SeVI_15, SeVI_45, SeVI_135). Average Values for n=3 Independent Replicates, each Analysed in Triplicate, are reported. Standard Deviations in Brackets. Different Letters within each Column Indicate Statistically Significant Differences at P < 0.05. Treatments

TChlC

Pigments (mg g-1 DM) TCC TChlC/TCC ratio

Se_0

17.2 (0.48) b

5.80 (0.09) a

2.98 (0.23) b


20.7 (0.50) a 20.7 (0.36) a 13.1 (0.29) c 12.5 (0.44) c

6.07 (0.14) a 4.83 (0.23) b 4.55 (0.25) b 3.90 (0.17) c

3.41 (0.01) b 4.31 (0.26) a 2.89 (0.13) ce 3.21 (0.09) c


16.2 (0.55) b 13.4 (0.11) c 7.85 (0.56) d

5.65 (0.13) a 4.96 (0.29) b 3.01 (0.18) d

2.87 (0.11) ce 2.73 (0.15) de 2.60 (0.04) e



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Table 5. Soluble Free, Soluble Conjugated and Bound Phenolic Acids (µg g-1 DM) in Shoots of 10-Day Old Rice Sprouts Obtained with Distilled Water (Se_0) or at 15, 45, 135 and 405 mg L-1 of Sodium Selenite (SeIV_15, SeIV_45, SeIV_135 and SeIV_405) and Sodium Selenate (SeVI_15, SeVI_45, SeVI_135 and SeVI_405). Average Values for n=3 Independent Replicates, each Analysed in Triplicate, are reported. Standard Deviations in Brackets. Different Letters within each Column and each PA Form (Free, Conjugated, Bound) Indicate Statistically Significant Differences at P < 0.05. Phenolic acids (μg g-1 DM)

Treatments (mg Se L-1)

Hydroxybenzoic acids

Hydroxycinnamic acids

Total

GA

α-RA

GeA

VA

SaA

p-CA

FA

SiA

o-CA

Free Se_0

30.4 (1.3) de

nd

nd

nd

nd

nq

nq

nq

nq

SeIV_15

29.9 (0.7) e

nd

nd

nq

nq

nq

nq

nq

nq

29.9 (0.7)a

SeIV_45 SeIV_135

34.5 (1.4) c 36.9 (0.7) b

nd nd

nd nd

nq nq

nq 52.1(1.3) b

nq nq

nq nq

nq nq

nq 27.7 (1.3) a

34.5 (1.4)b 117 (2.0)f

SeIV_405

37.0 (2.1) b

nd

nd

nq

58.9 (1.4) a

nq

147 (0.7)

nq

27.4 (1.0) a

271 (2.7)g

SeVI_15 SeVI_45

39.5 (0.7) a 32.0 (0.7) d

nd nd

nd nd

nd nd

29.4 (0.7) d 29.9 (1.42) d

nd nd

nq nq

nq nd

nq nq

68.9 (1.0)d 61.8 (1.6)c

SeVI_135 Conjugated Se_0

30.0 (0.6) e

nd

nd

nd

46.6 (1.14) c

nd

nq

nd

nq

76.1 (1.3)e

5.2 (0.7) d

nd

nq

nd

26.5 (0.7) h

nd

nd

nd

nd

31.7 (0.9)a

SeIV_15 SeIV_45

nq nq

nd nd

53.6 (0.7) c 132 (4.7) b

nd 55.5 (2.0) c

350 (4.8) d 521 (9.5) b

nd nd

nd 27.8 (0.7) e

nd nd

nd nd

403 (4.8)b 737 (10.8)d

SeIV_135 SeIV_405

nq nq

nd nd

191 (9.2) a nq

82.4 (0.7) b 90.4 (2.1) a

536 (11.9) a 322 (9.6) e

257 (9.20) a nd

25.7 (0.7) e 36.3 (0.7) d

41.5 (0.7) a nd

nd nd

1133 (17.7)h 449 (9.8)c

SeVI_15

31.6 (0.7) b

nd

nq

32.3 (3.6) d

370 (4.3) c

nd

320 (2.9) b

nd

nd

754 (6.3)e

SeVI_45 SeVI_135 Bound

34.1 (0.7) a 22.7 (0.6) c

nd nd

191 (0.7) a 140 (1.1) b

35.5 (0.7) d 52.8 (0.6) c

87.4 (2.8) g 301 (8.00) f

118 (5.7) b nd

372 (2.8) a 298 (4.0) c

29.9 (1.4) b nd

nd nd

868 (7.1)g 815 (9.0)f

30.4 (1.3)a

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Se_0

25.2 (1.3) b

13.6 (0.7)

12.3 (0.7) b

nd

nd

nq

13.6 (0.7) b

nd

nd

64.6 (1.7)e

SeIV_15 SeIV_45

13.6 (0.7) c 27.8 (1.4) a

nq nd

nd 14.9 (1.4) a

nd nq

nd 12.9 (0.7) c

nd nq

15.6 (0.7) a 14.9 (0.7) a

nd nd

nd nd

29.2 (1.0)a 70.4 (2.2)f

SeIV_135 SeIV_405

nd 28.8 (1.4) a

nd nd

nd nq

nd nd

19.8 (1.3) a nd

nd nd

15.8 (0.7) a nd

nd nd

nd nd

35.6 (1.5)b 28.8 (1.4)a

SeVI_15

15.1 (0.7) c

nd

15.8 (2.2) a

nd

15.8 (1.3) bc

nd

nd

nd

nd

46.7 (2.6)d

SeVI_45 SeVI_135

14.9 (0.7) c 10.8 (0.6) c

nd nd

13.5 (1.4) ab 11.9 (0.6) b

nd nd

17.1 (1.4) b 16.5 (0.6) b

nq nq

nd nd

nd nd

nd nd

45.5 (2.1)d 39.2 (1.0)c

GA: gallic acid; α-RA: α-resorcylic acid; GeA: gentisic acid; VA: vanillic acid; SaA: salicylic acid; p-CA: p-coumaric acid; FA: ferulic acid; SiA: sinapic acid; o-CA: o-coumaric acid; nd: not detectable; nq: not quantifiable.

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Figure 1


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Selenium biofortification in rice (Oryza sativa L.) sprouting: effects on

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