Metabolite Profiling of Sugarcane Genotypes and Identification of

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Metabolite profiling of sugarcane genotypes and identification of flavonoid glycosides and phenolic acids Isabel D. Coutinho, John M. Baker, Jane L. Ward, Michael H. Beale, Silvana Creste, and Alberto José Cavalheiro J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.jafc.6b01210 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 11, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

1

Metabolite profiling of sugarcane genotypes and identification of

2

flavonoid glycosides and phenolic acids

3

Isabel D. Coutinho†*, John M. Baker‡, Jane L. Ward‡, Michael H.

4

Bealeb‡, Silvana Creste††, Alberto J. Cavalheiro†

5

†

6

Mesquita Filho”

7

14800-060, Araraquara, São Paulo, Brazil.

8

‡

9

Harpenden AL5 2JQ, United Kingdom.

Instituto de Química, Universidade Estadual Paulista “Julio de (UNESP), Rua Prof. Francisco Degni, 55, CEP

Plant Biology and Crop Science Department, Rothamsted Research,

10

††

11

Nogueira, km 321, CP 206, CEP 14032-800, Ribeirão Preto, Brazil.

Instituto Agronômico – Centro de Cana, Rodovia Antonio Duarte

12 13 14 15 16 17 18 19 20 21 1 ACS Paragon Plus Environment


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ABSTRACT: Sugarcane is an important agricultural crop in the

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economy of tropical regions, and Brazil has the largest cultivated

24

acreage in the world. Sugarcane accumulates high levels of sucrose in

25

its stalks. Other compounds produced by sugarcane are currently not

26

of economic importance. To explore potential co-products we have

27

studied the chemical diversity of sugarcane genotypes, via metabolite

28

profiling of leaves by NMR and LC-DAD-MS. Metabolites were

29

identified via in-house and public databases. From analysis of 20

30

HPLC-fractionated extracts, LC-DAD-MS detected 144 metabolites,

31

of which 56 were identified (MS-MS and 1H NMR), including 19

32

phenolics and 25 flavones, with a predominance of isomeric flavone

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C-glycosides. Multivariate analysis of the profiles from genotypes

34

utilized in Brazilian breeding programs revealed clustering according

35

to sugar, phenolic acid and flavone content.

36 37

KEYWORDS: sugarcane, amino acids, phenolic acid, flavones,

38

metabolite profiling

39 40 41

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INTRODUCTION

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The Saccharum genus, together with Sclerostachya, Narenga,

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Erianthus and Miscanthus genera constitute a closely-related inter-

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breeding group known as the ‘Saccharum Complex’.1 With the

46

exception of sugarcane, most genera belonging to this group generally

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have low sugar content and differing stalk morphology.2 Saccharum

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has unique physiology and produces extremely high biomass yields

49

while also accumulating high concentrations (> 600 mM) of sucrose

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in its culm.3 Modern sugarcane varieties are derived mainly from

51

interspecific crosses between S. officinarum L. and S. spontaneum.

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Hybridizations emerged as a solution to losses caused by diseases and

53

need to obtain more resistant plants.5

4,5

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Beyond being an established source of sugar, sugarcane is the

55

current benchmark first-generation feedstock for efficient biofuel

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production, as well as, animal feed, sugarcane spirit, sugarcane syrup,

57

and other products.

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metabolites from sugarcane leaf extracts were published in the 1950s.

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6-7

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genera from Saccharum Complex leading to the identification of eight

61

flavone C- and O-glycosides.8 In the last ten years, further studies

62

have reported the presence of flavones in Saccharum.9-16 In addition,

The first reports concerning secondary

Later, Williams et al carried out taxonomic characterization of



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phenolic compounds and triterpenes have also been described in

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sugarcane leaves. 17,20

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Despite the key role of sugarcane in the economy of Brazil, there

66

have been few comprehensive studies of the metabolite and genetic

67

diversity of the genotypes developed by Brazilian breeding programs

68

(IAC-IACSP, SP and RB). The genetic variability of modern varieties

69

has been investigated by target region amplification polymorphism

70

(TRAP),21 whilst tentative metabotype discrimination amongst RB

71

varieties has been proposed by 1H NMR in solution and in solid

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matrices by HR-MAS (High Resolution Magic Angle Spinning),22 but

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this study focused on the content of primary metabolites, mainly

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sucrose.

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In this paper, we provide a phytochemical catalogue of sugarcane

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leaves derived from metabolite screening of sugarcane genotypes. The

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applicability of identified metabolite is demonstrated in a study of

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metabolomic profiling of thirteen sugarcane genotypes by 1H NMR

79

and LC-DAD to provide metabolite markers for future breeding

80

programs.

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MATERIALS AND METHODS

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Plant material. Sugarcane genotypes (RB966928, IACSP955000,

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IACSP933046, IACSP974039, SP803280, RB92579, RB835486, 4 ACS Paragon Plus Environment

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IAC912218, IAC911099, IACSP962042, IACSP977569, RB867515

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and CB49260) belong at different Brazilian breeding programs: IAC

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(Instituto

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(República do Brasil) and CB (Campos Brasil) were cultivated at

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greenhouse, in Ribeirão Preto – SP, Brazil (21o11’ S, 47o48’ W) in 50

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litre pots containing a 3:1:1 mixture of soil, sand, and pine and

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coconut bark substrate (Tropstrato), and fertilized according to Van

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Raij et al. (1996).23 Leaf samples (leaf +1) of first-cut plants were

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collected between 8:30 and 9:00 a.m. when plants were nine months

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old and used for metabolomics screening. For phytochemical analysis,

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leaves of seedling IAC955000 were collected with two months age.

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After harvest, plant material was immediately frozen under liquid

96

nitrogen and stored at -80 °C. Prior to extraction, the samples were

97

lyophilized and milled in a cryogenic mill using a first step for sample

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freezing (1 min) followed by 1 cycle with 1 stage of pulverization and

99

cooling to obtain particles smaller than 60 µm.

Agronômico

de

Campinas),

SP

(Copersucar),

RB

100

Extracts for metabolite isolation. An extract was obtained from 500

101

mg of leaves IAC95 5000 genotype (EMI) and 5 mL of

102

water:methanol (80:20 v/v) employing a methodology developed by

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Ward et al, 2003 and Lewis et al, 2004.24,25 The samples were mixed

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using a bench top whirlimix for 30 seconds. After heating at 50 °C in 5 ACS Paragon Plus Environment


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a water bath for 10 minutes, the plant residue was pelleted with a

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bench top centrifuge for 5 minutes. To arrest hydrolytic enzyme

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activities, the resulting supernatant was heated at 90 °C in water bath

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for 2 minutes. The tube was cooled for 30 minutes, and concentrated

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under vacuum to 1.0 mL. Metabolites were fractionated by

110

preparative HPLC (below).

111

Extracts for metabolite profiling. To 50 mg of the sugarcane

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genotypes milled leaves was added 750 µL of D2O:CD3OD (80:20

113

v/v)

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trimethylsilylpropionic acid). The contents of the tube were mixed

115

using a bench top whirlimix for 30 seconds, then, heated at 50 °C in a

116

water bath for 10 minutes. The samples were then centrifuged for 5

117

minutes. Supernatant (750 µL) was heated at 90 °C in a water bath for

118

2 minutes and then cooled to 4 °C for 30 minutes. Supernatant (200

119

µL) were transferred to a 3 mm NMR tube for NMR analysis. A

120

further aliquot of supernatant (400 µL) was collected and diluted with

121

H2O:CH3OH 80:20 v/v (200 µL) for LC-DAD analysis. The extracts

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were prepared in duplicate.

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Preparative High Performance Liquid Chromatography. The

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EMI extract was fractionated by on an Agilent 1100 HPLC system

125

equipped with a G1311A Quaternary pump, G1315B diode array

containing

0.02%

w/v

TSP-d4

(sodium

salt

of


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detector, G1313A autosampler, G13161A column oven, ISCO

127

fraction collector (model FOXY JR) and Agilent Chemstation

128

(RevA08) software, using an Ascentis C18 column (250 x 5 mm i.d.,

129

5 µm). The gradient of elution was performed with water/0.1% formic

130

acid (A) and acetonitrile/0.1% formic acid (B) under the following

131

conditions: 0 min, 5% B; 60 min, 29%B; 63 min, 100%B, 66 min,

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100%, 69 min, 5% B, 80 min, 5%B. Flow rate at 1.0 mL/min and

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injection volume of 100 µL. The EMI were fractionated in 60 one

134

minute (1 mL) fractions, collected from 0-60 minutes. This procedure

135

was repeated 8 times. Equivalent fractions were combined and dried

136

in a vacuum concentrator. Each combined fraction was solubilized in

137

650 µL of D2O:CD3OD (80:20 v/v) containing 0.01% w/v TSP-d4,

138

where 600 µL were transferred to a 5 mm NMR tube for NMR

139

analysis. Then, the ratio of the area of resonance of TSP at δ 0.00 was

140

used to estimate the relative concentration of metabolites that did not

141

showed overlapping of the signals in 1H NMR spectra. This approach

142

was employed to get the general level of metabolites isolated. A

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further aliquot of 20 µL was collected and diluted with H2O:CH3OH

144

80:20 v/v (200 µL) for LC-DAD-MS/MS analysis.

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LC-DAD-MS system used for EMI fractions analysis. All fractions

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collected from preparative HPLC were analysed by LC-DAD-MS 7 ACS Paragon Plus Environment


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using the LC-(DAD)-LTQ-Orbitrap Elite system consisting of a

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Dionex ULTIMATE 3000 UHPLC equipped with a LPG-3400RS

149

quaternary pump, a DAD-3000 photodiode array detector, a

150

WPS3000TRS thermostated autosampler and a TCC-3000 RS column

151

compartment, coupled to a Thermo LTQ-Orbitrap Elite, with a heated

152

ESI source (Thermo Scientific, Hemel Hempstead, UK). UV spectra

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were acquired from 230-400 nm. Mass spectra were acquired in

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negative and positive modes with a resolution of 120,000 over m/z

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range of 50-1500, in separated runs. Operating parameters were as

156

follow: source voltage, 2.5 kV, sheath gas, 35 (arbitrary units);

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auxiliary gas, 10 (arbitrary units); sweep gas, 0.0 (arbitrary units); and

158

capillary temperature, 350 °C. Default values were used for most

159

other acquisition parameters. Automatic MS-MS was performed on

160

the three most abundant ions of each scan, with the Orbitrap

161

resolution set at 15000 over m/z 50+. An isolation width of m/z 2 was

162

used and precursors were fragmented by high-energy C-trap

163

dissociation (HCD) with normalized collision energy of 65, and an

164

activation time of 0.1 ms. The maximum injection time for the FT was

165

set to 200 ms for MS and MSn mode. The data analyses were

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performed using XCalibur software. The chromatographic runs were

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performed using a Hypersil gold column (1.9 µm, 30 x 2.1 mm i.d., 8 ACS Paragon Plus Environment

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Thermo, Hemel Hempstead, UK) which was maintained at 25°C and a

169

solvent system of water/0.1% formic acid (A) and acetonitrile/0.1%

170

formic acid (B) (Fisher Scientific). Separation was carried out over

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24.5 minutes under the following gradient conditions: 0 min, 5% B;

172

22 min, 31.6% B; 23 min, 100% B, 24.6 min, 100%. Flow rate at 0.3

173

mL/min and injection volume of 10 µL. For metabolic profiling by

174

LC-DAD, the same chromatographic conditions described above were

175

used, except the column used was a Kinetex® C-18 column (2.6 µm,

176

50 x 2.1 mm i.d., Phenomenex).

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A second LC-ESI-MS system was used to confirm and

178

characterize compounds with m/z 535. This system was an Agilent

179

HPLC with a 1200 quaternary pump, a 1200 autosampler coupled to a

180

mass spectrometer (MS) with hybrid quadrupole/linear ion trap (3200

181

QTRAP, AB SCIEX) analyzer. Ionization was achieved by

182

electrospray (ESI) in negative mode.

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IonSpray voltage (IS): - 4500.0 V, declustering potential (DP): -

184

4500.0 V, entrance potential (EP): - 10.0 V, cell entrance potential

185

(CEP): - 31.6 V. The following source parameters were also

186

employed: temperature: 700˚C, Gas 1, Gas 2: 50 psi and Curtain Gas

187

(CUR): 10 psi. Initially, mass spectrometry (MS) analyses were

188

performed in full scan mode for compounds detection. MS2

The parameters used were:



were

also

performed

for

molecular

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experiments

structure

190

confirmation, using collision energy (CE) of 45.0 and 30.0 V and

191

collision energy spread 15 V. The chromatography condition included

192

a Luna® C-18 column (5.0 µm, 150 x 4.5 mm i.d., Phenomenex),

193

maintained at 40 °C and eluted with water/0.1% formic acid (A) and

194

acetonitrile/0.1% formic acid (B), in gradient mode: 0min, 5% B;

195

50min, 29%B; 53min, 100%B, 56 min, 100%, 59min, 5% B, 70min,

196

5%B. Flow rate at 1 mL.min-1 and injection volume of 20 µL.

197

Nuclear Magnetic Resonance. The fractions collected by preparative

198

HPLC were analysed by 1H-NMR. The spectra were acquired under a

199

temperature of 300 K on an Avance 600 spectrometer (Bruker

200

Biospin, Coventry, UK) operating at 600.0528 MHz using a 5 mm

201

SEI probe. The residual HOD signal was suppressed by pre-saturation

202

during 5 s relaxation delay. Each spectrum consisted of 128 scans of

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64 K data points with a spectral width of 12 ppm. The spectra from

204

sugarcane genotypes leaves extracts were acquired using on Avance

205

III (operating at 600.1298 MHz, equipped with a BBFO-Z plus

206

SmartProbe Broadband Observe a 5mm). The noesygppr1d pulse

207

sequence was employed to suppress of residual HOD signal. The

208

proton spectra were acquired with a 4.6 s presaturation delay,

209

acquisition time 2.72 s (64 k points), accumulation of 256 transients 10 ACS Paragon Plus Environment

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and spectral width of 20 ppm. All the spectra FIDs were automatically

211

Fourier transformed after the application of an exponential window

212

function with a line broadening of 0.3 Hz. Phasing and baseline

213

correction were carried out within the instrument software. 1H NMR

214

chemical shifts were referenced to TSP-d4 at δ 0.00.

215

Data analysis. The LC-DAD data were reduced to ASCII files. Each

216

dataset was arranged in a XIxJ matrix, where I corresponded to rows

217

(26 samples) and J corresponded to columns (2700 variables). The

218

variables comprised the absorbance values at 350 nm (in mV), which

219

were recorded every 640 ms during the chromatographic run. The 1H

220

NMR spectra were automatically reduced to ASCII files using AMIX

221

(3.7, Bruker Biospin). Spectral intensities were reduced to integrated

222

regions of equal with (0.05 ppm) corresponding to the region of -0.50

223

to 10.00 ppm and the dataset was arranged in a X26x203 matrix. The

224

region corresponding to residual signal of water was excluded. The

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data preprocessing and Principal Component Analysis (PCA) from 1H

226

NMR and LC-DAD data were performed using Matlab 7.12.0

227

(MathWorks Co., Natick, MA).

228

RESULTS

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General profile. The EMI was submitted to initial screening

230

employing LC-DAD-MS and 1H NMR. To aid the identification of 11 ACS Paragon Plus Environment


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the metabolites the analytical method was scaled-up to a semi-

232

preparative chomatographic method, which was used to separate the

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metabolites into 60 one-minute fractions. All the data obtained were

234

annotated and the compounds characterized using the following

235

identification strategies: і) query the 1H NMR spectra and molecular

236

formulae generated from HR-MS (High Resolution – Mass

237

Spectrometry) using “in house” and public plant natural product

238

databases; іі) confirmation of annotation by UV spectra, MS/MS

239

fragmentation and further

240

compounds.

1

H NMR investigation of purified

241

This strategy provided the identification of 56 metabolites from

242

sugarcane leaves. Of these, sugars (1), two organic acids (2-3) and

243

nine amino acids (4-12) were identified employing an “in house” 1H

244

NMR

245

hydroxycinamic acids derivatives and twenty-five flavones were

246

identified, of which 28.0% had the structure confirmed by MS,

247

MS/MS, UV and 1H NMR data and 72.0% were assigned solely from

248

MS, MS/MS and UV spectra (Figure 1).

249

Diode-array detector (DAD). The most single, useful and versatile

250

detector in modern LC is the diode-array detector.26 The UV/Vis

251

spectrum provided by DAD enables the detection of a broad range of

database.

Five

benzoic

acid

derivatives,

eighteen


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metabolite types, and provides data on the classes of metabolite,

253

through characteristic spectra and absorbance maxima. The LC-UV

254

chromatogram at 320 nm of the sugarcane leaf extract is illustrated in

255

Figure 2, showing the UV spectra characteristic of the main

256

compound classes identified in this work.

257

The amino acids phenylalanine and tryptophan showed λmax of 258

258

and 279 nm, respectively. An intense band due to π- π* transition is

259

characteristic of aromatic compounds and a second aromatic band of

260

low intensity appears at 255-280 nm in the most simple benzene

261

derivatives.27 The identified benzoic acid derivatives showed λmax

262

around 298 nm (Figure 2, BA), i.e. gentisic acid glycoside, while the

263

phenylpropanoid derivatives show a characteristic absorbance at 300-

264

330 nm corresponding to cinnamoyl systems. The aldaric acid

265

derivatives were identified linked to different substitution pattern and

266

showed absorbance maximum in range 280-308 nm (Figure 2, HA).

267

The flavonoids show a first band in the region 240-285 nm, due to

268

the A-ring and a second band, in the region 300-550 nm, which is

269

affected by the substitution pattern and conjugation of the C-ring.28

270

These features allowed the distinction of apigenin (band B at λmax 336

271

nm) from tricin (band B at λmax 351 nm) derivatives. Small shifts in

272

λmax distinguish different types of flavones, such as, apigenin, luteolin, 13 ACS Paragon Plus Environment


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diosmetin and tricin derivatives, which showed a small bathochromic

274

shift due the substituent pattern on the B-ring.

275

Nuclear Magnetic Resonance and Mass spectrometry. The

276

combined use of

277

requirement to improve the accuracy of the identification of the

278

compounds. However, due the lower sensitivity of 1H NMR only

279

major metabolites were confirmed. In some cases, only the diagnostic

280

1

281

Supporting Information) and this feature was used to calculate the

282

relative molar ratio of some metabolites, such as, sucrose (3910 µM),

283

phenylalanine (530 µM), apigenin-6-C-arabinosyl-8-C-glucoside (20

284

µM), tricin-7-O-α-L-rhamnosyl-glucuronide (80 µM), tricin-7-O-

285

glucuronide-sulfate (70 µM) and trans-3-feruloylquinic acid (50 µM)

286

relative to TSP as internal reference. Through a semi-quantitative

287

approach, we achieved that the concentration of secondary

288

metabolites present is sugarcane leaves is less than 2.0 % compared to

289

sucrose content.

1

H NMR and LC-MS/MS was fundamental

H NMR signals were used for identification (See Tables 1 and 2 in

290

The primary metabolites 2, 3, 4 and 11 eluted at 1.41, 1.41, 2.05

291

and 3.84 minutes were also detected by LC-MS/MS. The isomers

292

trans aconitic acid and cis aconitic acid were identified via a

293

pseudomolecular ion m/z 173 [M-H]-, while the phenylalanine and 14 ACS Paragon Plus Environment

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tryptophan were detected in positive mode via pseudomolecular ions

295

at m/z 166 and 188 [M+H]+, respectively.

296

The total ion chromatogram obtained by LC-DAD-MS is shown in

297

Figure 3. Accurate mass measurement provided by the Orbitrap was

298

used for generate a set of possible molecular formulae. The set of

299

molecular formulae generated by the XCalibur software was first

300

checked against an in-house databank. If the molecular formula

301

generated were not found in the sugarcane databank, the Reaxys

302

database was then consulted to propose structures.

303

spectra were used to confirm the identity of these proposed structures.

304

Three dihydroxybenzoic acid glycoside isomers (14, 15, 16) and

305

one simple benzoic acids glycoside (hydroxy benzoic acid glycoside)

306

were easily located in the extracted ion chromatograms of their

307

pseudomolecular ions m/z 315 [M-H]- (C13H16O9) and m/z 299 [M-H]-

308

(C13H15O8),

309

dihydroxybenzoic acid glycoside isomers was possible by evaluating

310

the from abundance of radical ions as a result of the hydrogen

311

bonding.

312

product ion m/z 137 in MS/MS spectra resulting from the loss of the

313

glucose (162 Da) moiety.

30

respectively

(Figure

S1).

The

29

Then, MS/MS

identification

of

The hydroxyl-benzoic acid glycoside (23) showed a



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314

The caffeic acid derivatives (17, 21, 24, 25, 27) showed m/z 353

315

[M-H]- (C16H18O9) ion in negative mode and gave specific

316

fragmentation patterns for each compound.31,32,33 In addition to the

317

UV, MS and MS/MS data, the compounds 16, 24, 27 were confirmed

318

by 1H NMR (Table 3).

319

In addition, we also identified aldaric acid derivatives including

320

coumaroyl, benzoyl and syringyl moieties. Aldaric acid is a term used

321

to describe a family of isomeric dicarboxylic acids, derived from

322

aldoses, the most common forms being glucaric and manaric acid.

323

A glucaric acid form was established by 1H NMR and a fragmentation

324

scheme has been proposed (Figure S8). As reported in the literature

325

using the MS,

326

feruloyl, benzoyl and coumaroylsyringil) could not be distinguished

327

by MS. Therefore, compounds 17 and 19 were identified as

328

coumaroyl esters of a glucaric acid, 29 as a feruloyl ester of a glucaric

329

acid, 20, 22 and 28 as benzoyl esters of a glucaric acid and 32 and 36

330

as coumaroylsyringyl esters of a glucaric acid. MS/MS of product

331

ions of the glucaric acid derivatives often generated the fragments

332

ions m/z 85 (bp), 173 and 209 (Figures S2-S9).

35,36

34

the exact position of substituents (coumaroyl and

333

A great diversity of glucose, arabinose, rhamnose and glucuronic

334

acid C-glycosides of apigenin, luteolin, and diosmetin were observed 16 ACS Paragon Plus Environment

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as well as O-glucosylated tricin derivatives (Figure 1). The C-

336

glucosylated flavone shows a fragmentation patterns different to O-

337

glucosylated flavone, and that the O-glycoside flavone has mass

338

spectra with the base peak corresponding to product ion of aglycone,

339

while the flavones C-glycosides presented loss of 60, 90, 120 and 150

340

Da corresponding the fragmentation of sugar moiety.

341

features were important to distinguish them. In addition, it was

342

possible to identify flavones C-glycosides isomers based on intensity

343

differences of fragment ions referent to cleavage of sugar unit binding

344

to 6 or 8-C. Detailed information about identification of flavones are

345

available in Supporting Information (Figures S10-S15).

346

Data analysis using multivariate statistics. Application of PCA to

347

revealed differences between the spectra of sugarcane genotypes

348

extracts. As shown in Figure 4 (A), there is a clear discrimination

349

among the genotypes of IAC, RB and CB sugarcane. This separation

350

took place in the first two principal components, which cumulatively

351

accounts for 80% of explained variation. The separation between RB

352

varieties and the varieties IAC, SP and CB was easily achieved on the

353

basis of the scores of principal component 1 (PC1) and principal

354

component 2 (PC2). IAC and SP genotypes were clustered at negative

355

PC1 and PC2, CB was located at positive PC1 and negative PC2,

37, 38

These



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356

while the RB varieties were quite well separated from them. It was

357

possible to determine the variable importance by analysing correlation

358

of each variable with scores plot. The loading plot of all 1H NMR

359

evaluated signals is shown in Figure 4 (B). It showed that mainly IAC

360

and SP genotypes contain a much higher level of ferulic acid and

361

glucose.

362

Once the samples were grouped according to the major metabolites

363

on the basis of 1H NMR data, we applied PCA to LC-DAD data to

364

provide a chemical classification based on minor metabolites

365

(phenolic acids and flavones) present in sugarcane genotypes leaves

366

and not detected by 1H NMR spectroscopy. Figure 4 (C) shows the

367

comparative PCA analysis of sugarcane genotypes (1-13) from LC-

368

DAD data. According to the PCA score plot, component one explains

369

47.97% of the variation and component two accounts for 14.01% of

370

the variation, showing a clear separation between three distinct groups

371

of genotypes. The IAC genotypes were clearly separated from RB

372

and CB genotypes, except for RB92579 (6) which clustered between

373

IAC genotypes indicating different metabolic profile among genetic

374

breeding program. The metabolites responsible for the separation in

375

the scores plot are displayed in loadings plots (Figure 4, D). Loadings

376

1 shows the metabolites responsible for separation between IAC and 18 ACS Paragon Plus Environment

Page 19 of 45


377

RB/CB genotypes as given by PCA component and loading 1. The

378

genotypes IACSP962042 and IACSP977569 (10 and 11) have higher

379

concentration of chlorogenic acids (HA) than RB varieties (12 and 1).

380

While another IAC varieties, located at PC1 negative and PC2

381

positive showed higher correlation of apigenin, luteolin and tricin

382

derivatives.

383

DISCUSSION

384

All the fractions obtained from preparative scale chromatography

385

were analyzed by LC-DAD-MS/MS and 1H NMR, and the data were

386

used to perform metabolomics screening of sugarcane leaves. The

387

combined use of chromatographic separation and two orthogonal high

388

resolution spectroscopy techniques resulted to be particularly suited

389

for accurate identification of metabolites. In addition, the results from

390

metabolomics screening showed that 1H NMR profiles were not

391

optimal for identification of discriminatory secondary metabolites

392

(except for ferulic acid, an abundant metabolite), as the profiles are

393

dominated by the sucrose levels. However, LC-DAD technique

394

showed appropriate for metabolomics analysis. As a result, PCA from

395

LC-DAD data was able to discriminate sugarcane genotypes due to

396

variability of flavones and phenolic acids.



Page 20 of 45

397

Among the identified metabolites, we observed the presence of

398

quinic acid esterified with coumaroyl, caffeic and ferulic acid units

399

and glycosylated protocatechuic acid in sugarcane seedlings.

400

Furthermore, we detected free ferulic acid in mature sugarcane

401

genotypes. Ferulic acid is an important molecular marker, because it

402

contributes to the formation of lignin in mature cane, which varies

403

among genotypes, cell types and among tissues in the same plant. 39,40

404

Since oxidative coupling of coniferyl alcohol (formed from reduction

405

of ferulic acid), catalyzed by peroxidase enzymes, leads to the

406

formation of lignin,

407

SP803280, IACSP962042 and RB92579 have different regulatory

408

mechanisms for lignin synthesis compared to other genotypes.

40

we can infer that genotypes IACSP955000,

409

Coumaroyl and caffeic acids were found esterified with the less

410

common aldaric acid. The enzyme chlorogenic acid: glucaric O-

411

caffeoyltransferase has been identified in Lycopersicon esculentum

412

and catalyzes the transfer of caffeic acid from 5-O-caffeoylquinic acid

413

to aldaric acid.

414

have been identified in sugarcane and Poaceae family, although, it

415

was previously identified in Smallanthus sonchifolius

416

microphylla 42 and Vigna sinensis 43.

41

This is the first time that aldaric acid derivatives

41

, Berberis


Page 21 of 45

417


We

also

identified

both

the

hydroxybenzoic

acid

and

418

dihydroxybenzoic connected to sugar moieties, including gentisic acid

419

5-O-β-glucoside. Gentisic acid is a metabolite analogous to salicylic

420

acid, and it has been considered as signaling molecule in the defense

421

response of plants to pathogens.

422

gentisic acid was reported following systemic infections. In tomato,

423

gentisic acid acts as a pathogen signaling in the same way as salicylic

424

acid activating defense genes

425

compounds in sugarcane may be important in future metabolomics

426

studies in seedlings under biotic stress.

45

44

Accumulation of high levels of

. Therefore, identification of these

427

Beyond phenolic acids, we identified flavonoids belonging to the

428

flavones class. However, intermediates of their metabolic pathway,

429

such as chalcones and flavanones were not detected. Among the

430

flavones, we identified C-glycosylated apigenin, luteolin and

431

diosmetin and O-glycosylated tricin. These flavones profiles are

432

characteristic of the grasses species. Flavones 35, 36, 38, 39, 43, 47,

433

51 and 54 were reported in S. officinarum, S. edule, S. robustum, S.

434

sinensis and S. spontaneum.8,46 The flavones 32, 33, 37, 42, 44, 46

435

and 48 have been described in Poaceae, while the metabolite 52 is

436

reported for the first time. The presence of sulfate conjugate of tricin-

437

glucoside in sugarcane was reported by Williams, Harbone and Smith, 21 ACS Paragon Plus Environment


Page 22 of 45

438

1974,8 but in that case the sugar moiety was identified as glucose, and

439

not as glucuronic acid, as in our work.

440

Different functions have been attributed to flavonoid C-glycosides,

441

such as antioxidants, insect, feeding attractants, antimicrobial agents,

442

promoters of mycorrhizal symbioses and UV-absorbing pigments. In

443

many cases, these functions require high local concentration and

444

many of these compounds are harmful to the plant.46 Flavone C-

445

glycosides have already been described as phytoalexins after their

446

discovery in cucumbers infected with powdery mildew fungus. 47

447

Based on our work, the genotypes IACSP95500, IAC912218 and

448

RB835486 are important source of flavones, mainly luteolin and tricin

449

derivatives. It is worth noting that these genotypes showed resistance

450

to mosaic virus.48 IACSP95500 showed lowest infestation intensity

451

rates of sugarcane borer Diatraea saccharalis49 and resistance to

452

Puccinia kuehnii16, which could be associated to high content of

453

flavones C-glycosides.

454

Besides a physiological function in the plant, the flavones

455

identified in this work have well known antioxidant activity, mainly

456

apigenin and luteolin, which contain hydroxyl groups in orto in ring

457

A/B showed antioxidant properties at low concentrations and

458

presented therapeutic activity, such as, antimalarial, antimicrobial, 22 ACS Paragon Plus Environment

Page 23 of 45


459

diabetes and antioxidant.50,

460

flavonoids present in edible plants and in plants used in traditional

461

medicine to treat a wide variety of pathologies. This flavonoid has

462

cancer chemopreventive and chemotherapeutic potential.52 Tricin

463

derivatives have been described as potential cyclooxygenase 2

464

inhibitor-dependent anti-human cytomegalovirus activity

465

inflammatory action in human peripheral blood mononuclear cells

466

Taking this into account, we suggest that genotypes IACSP95500,

467

IAC912218 and RB835486 have potential for genetic improvement of

468

Sugarcane as a source of flavones, mainly luteolin and tricin

469

derivatives.

470

By

combining

the

51

Luteolin is one of the most common

detailed

structural

53

and anti-

information

54

.

and

471

metabolomics screening, we provided a good strategy to select

472

biomarkers and structurally identify metabolites, in accordance with

473

the minimum reporting requirements for metabolite identification. 55,56

474

In addition, our work may be integrated to transcriptomic and

475

proteomic data in future studies to understand how biotic and abiotic

476

stresses are regulated by metabolites or to select potential genotypes

477

for production of novel compounds beyond sucrose.

478 479 23 ACS Paragon Plus Environment


Page 24 of 45

480 481 482 483 484 485 486 487

ASSOCIATED CONTENT

488

Supporting Information

489

Detailed spectroscopic and structural information of metabolites

490

identified in this work is described in the supporting information. The

491

Extracted Ion Chromatogram of benzoic acid derivatives (S1) and p-

492

coumaroylglucarate acid isomer (S2), fragmentation proposal to p-

493

coumaroylglucarate acid (S3), Extracted Ion Chromatogram of

494

benzoylglucarate

495

coumaroylsyringylglucarate acid (S6), MS2 spectra of production ion

496

of coumaroylsyringylglucarate acid isomer I (S7), MS2 spectra of

497

production ion of coumaroylsyringylglucarate acid isomer II (S8),

498

proposed fragmentation scheme of syringylcoumaroylaldaric acid

499

isomer (S9), Extracted Ion Chromatogram of apigenin-glucosyl-

500

arabinoside isomers (S10), Proposal fragmentation scheme of luteolin

acid

(S4),

caffeoylglucarate

acid

(S5),


Page 25 of 45


501

6-C-arabinosyl-8-C-glucoside (S11), fragmentation pathways and

502

MS2 spectra relative to luteolin-8-C-arabinosyl-7-O-rhaminoside

503

tricin-7-O-rhamnosyl-glucuronide

504

scheme to tricin-7-O-rhaminosil-glucuronide (S13), tricin-4'- (O-

505

erythro/threo guaiacylglyceryl) ether-7-O-glucoside (S14), tricin-7-O-

506

glucuronidesulfate (S15). Typical maximum of absorption from

507

flavones derivatives isolated from sugarcane leaves (S16). Sugar,

508

organic acids and amino acids identified in the sugarcane leaves using

509

1

510

(8:2 v/v), (Table 1).

511

(Hz) of isolated secondary metabolites in D2O/CD3OD (8:2 v/v),

512

(Table 2). The Supporting Information is available free of charge via

513

the Internet at http://pubs.acs.org.

514

AUTHOR INFORMATION

515

Corresponding Author

516

*(I.D.C)

517

[email protected]

518

Funding

519

This work was supported by the National Counsel of Technological

520

and Scientific Development from the Brazilian Ministry of Education

521

and BBSRC – Biotechnology and Biological Science Research

(S12),

proposal

fragmentation

H NMR (δ chemical shift (coupling constants, Hz) in D2O/CD3OD 1

H chemical shifts (δ) and coupling constants

Phone

+(55)

16-3303-9010.

E.mail:



Page 26 of 45

522

Council. IDC scholarship from Science Without Borders Program of

523

CAPES, process n. BEX: 5606/13-6 from the Brazilian Ministry of

524

Education.

525

Notes

526

The authors declare no competing financial interest.

527

ACKNOWLEDGMENTS

528

The authors express their gratitude for the samples from IAC-Centre

529

of Cana.

530

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531

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sure? And how do your peers gauge your confidence.

744

Metabolomics 2014, 10, 350-353.


Page 37 of 45


FIGURES CAPTIONS Figure 1– Secondary metabolites identified in the hydro alcoholic extracts of sugarcane leaves. Figure 2 – UV Chromatogram, 320 nm, of the hydro alcoholic extract of the sugarcane leaves. BA: benzoic acid, HA: hydroxycinnamic acid, A: apigenin derivatives, T: tricin derivatives. Figure 3 – Total Ion Chromatogram (TIC) of hydro alcoholic extract of sugarcane leaves. Figure 4 – PCA performed from 1H NMR data (A and B) and HPLCDAD data (C and D). Scores and loadings plot PC1 X PC2. HA: hydroxycinnamic

acid;

A:

apigenin

derivatives;

L:

luteolin

derivatives; T: tricin derivatives. Genotypes: RB966928 (1), IACSP955000 (2), IACSP933046 (3), IACSP974039 (4), SP803280 (5), RB92579 (6), RB835486 (7), IAC912218 (8), IAC911099 (9), IACSP962042 (10), IACSP977569 (11), RB867515 (12) and CB49260 (13).



Page 38 of 45

TABLES Table 1 –Peak assignment of the sugarcane leaf hydro alcoholic extract using LC-DAD-MS/MS. Metabolites

RT

[M-H]-

UV

HCD

MS/MS (% intensities)

Formula

Error (ppm)

vanilic acid glucoside (13)

2.06

329.08755

-

21

167 (100)

C14H17O9

-0.78

gentisic acid 5-O-β-glucoside (14)

2.10

315.0722

298

20

108 (15), 109 (13), 150 (59), 153 (33), 315 (15)

C13H15O9

0.14

gentisic acid 2-O-β-glucoside (15)

2.68

315.0722

-

20

108 (1), 109 (8), 108(1), 109 (8), 315 (100 )

C13H15O9

0.56

trans 5-caffeoylquinic acid (16)

3.04

353.08777; 355.10266p

326

22

191 (100), 179 (80)

C16H17O9

1.06

p-coumaroylglucarate acid isomer I (17)

3.18

355.06696

308

23

85 (100), 147 (22), 163 (10), 173 (4), 191 (30), 209 (62)

C15H15O10

-0.31

protocatechuic acid 4-β-glucoside (18)

3.59

315.0723

-

20

109 (84), 153(100), 315 (30)

C13H15O9

-0.05

p-coumaroylglucarate acid isomer I (19)

4.18

355.06738

-

23

85 (100), 209 (88), 191 (38)

C15H15O10

0.96

benzoylglucarate isomer I (20)

4.41

313.05652

281

20

85 (100), 191 (22), 147 (27)

C13H13O9

1.05

coumaroylquinic acid (21)

4.77

337.11395

-

21

-

C13H21O9

-0.21

benzoylglucarate isomer II (22)

4.87

313.05630

-

20

85 (100), 111 (5), 121 (9), 129 (15), 147 (27), 191 (22)

C13H13O9

0.14

hydroxybenzoic-4-β-glucoside (23)

5.26

299.07721

-

19

93 (15), 135 (5), 137 (100), 299 (10)

C13H15O8

-0.1

trans-3-caffeoylquinic acid (24)

5.84

353.08760

324

22

161 (2), 173 (1), 179 (6), 191 (100)

C16H17O9

-0.72

trans-3-feruloylquinic acid (25)

6.5

367.10345

324

23

173 (5), 191 (4), 193 (100), 367(1)

C17H19O9

-0.02


Page 39 of 45


Metabolites

RT

[M-H]-

UV

HCD


Formula

Error (ppm)

p-coumaroylglucoside acid (26)

6.52

325.09274

283

21

163 (100)

C15H17O8

-0.34

trans 4-caffeoylquinic acid (27)

6.64

353.08762

-

22

161 (2), 173 (100), 179 (81), 191 (43), 353 (4)

C16H17O9

-0.53

benzoylglucarate isomer III (28)

6.98

313.05634

277

20

85 (100), 129 (190), 147 (32), 191 (21)

C13H13O9

1.05

caffeoylglucarate acid isomer (29)

8.94

371.06204

281

24

111 (100), 173 (30), 85 ( 24)

C15H15O11

-0.51

luteolin-6-C-arabinosyl-8-C-glucoside (30)

9.80

579.13531

271/347

37

369 (84,25), 399 (100), 411 (8), 429 (29), 459 (34), 489 (100), 579 (49)

C26H27O15

-0.40

luteolin-6-C-glucosyl-8-C-arabinoside (31)

9.87

579.13513

271/347

37

459 (100), 399 (85), 369 (65), 489 (41)

C26H27O15

0.17

coumaroylsyringylglucarate acid isomer I (32)

10.21

535.10706; 581.14465p

296

34

85 (100), 129 (72), 147 (45), 191 (29), 209 (11), 163 (46)

C24H25O14

-1.17

luteolin-8-C-glucoside (33)

10.82

447.09338

270/347

29

327 (100), 357 (77), 429 (8), 447 (17)

C21H19O11

1.19

apigenin-6-C-arabinosil-8-C-glucoside (34)

10.95

563.14050; 565.15289p

271/336

36

353 (51), 383 (36), 443 (72), 473 (48), 545 ( 5), 563 (100 )

C26H27O14

-0.23

luteolin-6-C-glucoside (35)

11.06

447.09360

270/347

29

327 (100), 357 (35), 447 (6)

C21H19O11

0.43

coumaroylsyringylglucarate acid isomer II (36)

11.38

535.11359; 581.14345p

291

34

85 (100), 129 (71), 147 (47), 163 (47), 191 (28), 197 (55), 209 (11)

C24H25O14

-1.17

luteolin-6,8-C-diarabinoside (37)

11.52

549.1223

279/345

35

459 (100), 399 (82), 369 (55)

C25H25O14

0.99

apigenin-6-C-glucosyl-8-C-arabinoside (38)

11.58

563.14868

271/335

36

443 (83), 353 (43), 473 (51), 383 (26)

C26H27O14

-0.24

apigenin-6,8-C-diglucoside (39)

11.6

593.15137

269/338

38

473 (100), 357 (32), 429 (35), 309 (25)

C27H29O15

1.27

apigenin-6-C-glucosylarabinoside (40)

12.29

563.13901

271/334

36

C26H27O14

0.002

293,046(100) 311,057(14,78) 341,067(11,29)



Metabolites

RT

[M-H]-

UV

HCD


Page 40 of 45

Formula

Error (ppm)

413,088(61,08) 563,141(13,78 ) apigenin-8-C-glucoside (41)

12.39

431.09875

269/334

28

311 (100), 341 (10), 431 (16)

C21H19O10

-0.951

apigenin-6-C-glucosylrhaminoside (42)

12.63

577.15601

271/339

37

293 (100), 311 (15), 323 (17), 341 (17), 413 (61), 577 (18)

C27H29O14

0.82

apigenin-6-C-glucoside (43)

12.64

431.09833

271/337

28

311 (100), 341 (39), 413 (2), 431 (16)

C21H19O10

-0.09

apigenin-6,8-C-diarabinoside (44)

12.67

533.12988

269/342

34

443 (61), 473 (32), 353 (26), 383 (25)

C25H25O13

0.91

tricin-O-(6’’-5’’’,6’’’-dimethoxycinamate) (45)

13.05

681.23969

-

44

329 (100)

C32H41O16

0.78

apigenin-8-C-glucosylrhaminoside (46)

13.14

577.15594

271/337

37

457 (58), 487 (26), 353 (31), 383 (20)

C27H29O14

0.76

diosmetin-6-C-glucoside (47)

13.38

461.10880; 463.12329p

271/346

29

341 (100), 371 (33), 443 (1), 461 (13)

C22H21O11

-1.16

luteolin-6-C-arabinoside (48)

14.15

417.08258

-

27

327 (100), 357 (32)

C20H17O10

0.96

luteolin 8-C-arabinosyl-7-O-rhamnoside (49)

14.31

563.14050

267/345

36

327 (100), 357 (55), 399 (56)

C26H27O14

0.42

tricin-7-O-glucuronide-sulfate (50)

14.54

585.06360

267/352

38

329 (7), 255 (100), 193 (14), 175 (72)

C23H22O16S

0.41

tricin-O-neohesperoside isomer (51)

14.65

637.24994

-

41

329 (100)

C29H33O16

-0.28

tricin-7-O-glucoside (52)

14.85

491.19211

-

31

329 (100)

C25H31O10

-0.33


15.05

637.17926

269/348

41

329 (100)

C29H33O16

-2.92

tricin-7-O-α-L-rhamnosyl-glucuronide (54)

15.77

651.15582; 653.17041p

269/351

42

329 (100)

C36H27O12

0.24


Page 41 of 45


Metabolites

RT

[M-H]-

UV

HCD


Formula

Error (ppm)


16.14

637.17413; 639.19128p

266/347

41

329 (100)

C29H33O16

-2.18

tricin-4'-(O-erythro or threo guaiacylglyceryl) ether glucoside (56)

17.7

687.19214

-

44

165 (38), 328 (6), 329 (100), 477 (6), 491 (11)

C33H35O16

-0.54

P: positive mode ionization; HCD: in eV; Formula: for detected [M-H]-



Page 42 of 45

FIGURES Figure 1 13 GluO

14 HO

O

O

Benzoic acid derivatives 15 18 HO O HO O OH

GluO

H3CO

23 GluO

O

OGlu HO

HO

OH

OH

OH

Hidroxicinnamic acid derivatives OH

HO CO2H O 16 HO

OH O 24 OHHO2C HO OH

O OH OH OH O

HO2C HO

OH OH O OCH3HO2C HO OH

O

OH

O

OH HO2C OH OH

O

25

O 27

OH

OH

OH

21

26

OH

OH HO

O OH

HO

OH O O

Glucaric acid derivatives O 17= coumaroylglucarate acid isomer I 19= coumaroylglucarate acid isomer II 20= benzoylglucarate acid isomer I O 22= benzoylglucarate acid isomer II 28= benzoylglucarate acid isomer III HO 29= f eruloylglucarate acid 32= coumaroylsyringylglucarate acid isomer I HO 36= coumaroylsyringylglucarate acid isomer II

HO OH caf f eoyl OH OH

OCH3 O HO OH coumaroyl

O HO

OH

O

OCH3 HO OCH3 OCH3 syringyl

OH

O glucaric acid

benzoyl

Flavone derivatives R4 R5

R3 R2O

O

R6

R1 OH O

Flavone derivatives

R1

R2

R3

R4

R5

R6

Apigenin-6-C-arabinosyl-8-C-glucoside (34)

Ara

H

Glu

H

OH

H

Apigenin-6-C-glucosyl-8-C-arabinoside (38)

Glu

H

Ara

H

OH

H

Apigenin-6,8-C-diglucoside (39)

Glu

H

Glu

H

OH

H

H

H

Glu-

H

OH

H

Apigenin-8-C-glucosylarabinoside (40) Apigenin-8-C-glucoside (41)

H

H

Glu

H

OH

H

Apigenin-6-C-glucosylrhamnoside (42)

Glu-

H

H

H

OH

H

Apigenin-6-C-glucoside (43)

Glu

H

H

H

OH

H

Apigenin-6,8-C-diarabinoside (44)

Ara

H

Ara

H

OH

H

Apigenin-8-C-glucosylrhamnoside (46)

H

H

Glu-

H

OH

H

Luteolin-6-C-arabinosyl-8-C-glucoside (30)

Ara

H

Glu

H

OH

OH

Luteolin-6-C-glicosyl-8-C-arabinoside (31)

Glu

H

Ara

H

OH

OH


Page 43 of 45


Luteolin-6-C-glucoside (33) Luteolin-8-C-glucoside (35) Luteolin 8,6-C-diarabinoside (37) Luteolin 8-C-arabinosyl-O-rhamnoside (49)

Glu

H

H

H

OH

OH

H

H

Glu

H

OH

OH

Ara

H

Ara

H

OH

OH

H

Rha

Ara

H

OH

OH

Glu

H

H

OCH3

OH

OH

Tricin-O-(6’’-p-methoxycinnamate)-glucoside

H

MetGlu

H

OCH3

OH

OCH3

Tricin-O-neohesperoside isomer (51, 53, 55)

H

Glu-

H

OCH3

OH

OCH3

Diosmetin-6-C-glucoside (47)

Tricin-7-O-glucoside (52)

H

Glu

H

OCH3

OH

OCH3

Tricin-7-O-α-rhamnosylglucuronide (54)

H

Rha-

H

OCH3

OH

OCH3

Tricin-7-O-glucuronidesulfate (50)

H

Glu

H

OCH3

OH

OCH3

Tricin-4’-(O-erythro/threo-guaiacylglyceryl)-

H

H

H

OCH3

Gua

OCH3

ether (56) Glu: glucose; Glc: glucuronide; Rha: rhamnose; Ara: Arabinose; Gua: guaiacylglyceryl ether; MetGlu: methoxycinnamate glucoside;

Figure 2



Page 44 of 45

Figure 3

Figure 4


Page 45 of 45


Toc Graphic


Metabolite Profiling of Sugarcane Genotypes and Identification of

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