Supplementary Material

Theoretical and Experimental Plant Physiology

DIMBOA levels in hexaploid Brazilian wheat are not associated with antibiosis against the cereal aphids Rhopalosiphum padi and Sitobion avenae

Jorge F. Pereira • André L. F. Sarria • Stephen J. Powers • Gudbjorg I. Aradottir • John C.

Caulfield • Janet Martin • Lesley E. Smart • John A. Pickett • Michael A. Birkett • Paulo R. V.

S. Pereira

J. F. Pereira ( ) • P. R. V. S. Pereira

Embrapa Trigo, Rodovia BR 285 km 294, Passo Fundo, RS, 99001-970, Brazil

E-mail: jorge.pereira@embrapa.br Phone: +55(32)3311-7400 Fax: +55(32)3311-7401

Present address:

J. F. Pereira

Embrapa Gado de Leite, Rua Eugênio do Nascimento 610, Juiz de Fora, MG, 36038-330,

Brazil

A. L. F. Sarria • G. I. Aradottir • J. C. Caulfield • J. Martin • L. E. Smart • J. A. Pickett • M. A.

Birkett

Biological Chemistry and Crop Protection Department, Rothamsted Research, West

Common, Harpenden, Hertfordshire, AL5 2JQ, UK

S. J. Powers

Computational and Systems Biology Department, Rothamsted Research, West Common,

Harpenden, Hertfordshire, AL5 2JQ, UK

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0 50 100 150 200 2500

500000

1000000

1500000

2000000

2500000

3000000

3500000

f(x) = 13135.9555434026 x − 24179.9126147631R² = 0.999076115972712

DIMBOA concentration (µgmL-1)

Area

(mAU

)

Fig. S1 Calibration curve obtained with DIMBOA standard solutions (n = 27)

Fig. S2 1H NMR (500 MHz, CD3OD) spectrum of the 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA).

2

N

O O

O

H3CO

OH

R2

5

78

8a

4a346

1

DIMBOA (R = H)

Fig. S3 13C NMR (125 MHz, CD3OD) spectrum of the 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA).

Fig. S4 1H-1H COSY (500 MHz, CD3OD) spectrum of the 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA).

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Fig. S5 HSQC (500 MHz, CD3OD) spectrum of the 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA).

Fig. S6 HMBC (500 MHz, CD3OD) spectrum of the 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA).

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Fig. S7 1H NMR (500 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

Fig. S8 13C NMR (125 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

5

N

O O

O

H3CO

OH

R2

5

78

8a

4a346

1

DIMBOA-Glc (R = Glc)

Fig. S9 Projection of the 13C NMR from HSQC (125 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

Fig. S10 Projection of the 13C NMR from HMBC (125 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

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Fig. S11 1H-1H COSY (500 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

Fig. S12 HSQC (500 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

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Fig. S13 HMBC (500 MHz, CD3OD) spectrum of the 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc).

m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

%

0

100 164

152

149113113101 131119

119127 136 143

155

158

234208

165

184180166

169173

194192

189198

206

232220

210 220

213222

224

246

237

241290262248 260

257 276264 274 286278 300293

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

100 152

123

122

113

149

131

164

454372

192

165

178

356235

194 234

220

248 354

341312274 302

404

440432

430

476

767

652536492

516

552630552 574 618 653

722700 732

789

790

849791821 850 970910887

924950 975 996

Fig. S14 Mass spectrum of DIMBOA (above) and DIMBOA-Glc (below) both in negative mode [M-H]-.

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Table S1 Mean concentration of DIMBOA in nine wheat cultivars at three time points. Data are presented on the natural log scale for statistical comparison of means using the maximum (assuming n = 3) standard error of the difference 0.1456 on 71 degrees of freedom invoking the least significant difference (LSD) value at the 5% level of significance: 0.2309.

Genotype 7 days 9 days 14 daysBR 32 2.392 0.295 0.007BRS 327 2.659 2.568 1.719BRS 328 2.716 1.809 1.373BRS Guabiju 1.667 0.808 -0.206BRS Guamirim 3.303 2.768 1.947BRS Parrudo 1.745 2.055 0.685PF869120 2.623 2.208 1.804Solstice 2.575 2.193 1.503BRS Timbaúva 3.418 2.452 1.836

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