Growth Rate Plots....1 A B Supplemental Figure 1. Growth Rate Plots. (A) Spaghetti plot showing no significant difference in growth rate between WT (black line), and PCBER-downregulated

1

A

B

Supplemental Figure 1. Growth Rate Plots. (A) Spaghetti plot showing no significant difference in growth rate between WT (black line), and PCBER-downregulated transgenic lines (colored lines) grown under greenhouse conditions. 31 WT plants and between 11 and 21 plants for each of the 4 transgenic lines were measured once a week for 15 weeks. Bars represent ±SD. (B) Spaghetti plot showing no significant difference in growth rate between WT and PCBER-downregulated poplars in semi-open (open–air cage-house) conditions. 11 WT plants and 7 plants for each of the 4 PCBER-downregulated transgenic lines were measured. Data show means ±SD.

Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260

2

WT PCBER-RNAi

Supplemental Figure 2. Electron Microscopy. Electron micrographs of sections through the stem of a WT and a PCBER-downregulated plant (PCBER-RNAi). V = xylem vessel, F = xylem fiber, R = xylem ray parenchyma. Bar = 5 μm.


3

Supplemental Figure 3. Cellulose quantification. Cellulose quantification showing no significant difference between WT and PCBER-downregulated poplars (Data show means ± SD , n = 5).


4

A

B

Supplemental Figure 4. Lignin Quantification. (A) Lignin quantification by the acetylbromide method; (Data show means ± SD, n = 5). (B) Lignin (aromatic region) comparative analysis by HSQC NMR showing no major compositional differences between WT and PCBER-downregulated plants.


5

Supplemental Figure 5. MS2 Fragmentation Pathways of the PCBER in Vivo Substrate. The structures of the ions resulting from collision-induced dissociation in negative-mode ionization are shown.


6


7

Supplemental Figure 6. Structures of Compounds Used in this Study. Compound 1 (DDC) is an 8–5 linked neolignan, compound 21 is an 8–8 linked lignan and compound 26 represents an 8–O–4 linked dilignol. For these compounds, the atoms are numbered and the A and B rings indicated. (+), PCBER reduces the compound and the product peak is less than 10% of the substrate peak; (++), PCBER reduces the compound and the product peak is more than 10% of the substrate peak; (-), PCBER does not reduce the compound. For reaction conditions, see Methods.


8

Supplemental Figure 7. MS Data for the PCBER Product. Accurate m/z data, MSn fragmentation spectra and proposed structure of the product of the reaction catalyzed by PCBER.


9

Supplemental Figure 8. MS2 Data of Scys(8–O–4)G. MS2 spectrum and gas phase fragmentation pathways of Scys(8–O–4)G, showing the structures of the ions resulting upon collision-induced dissociation in negative mode ionization.


10

Supplemental Figure 9. 2D NMR Spectra of Dilignol Glycerols. Dilignol glycerols are dihydroxylation products from DDC with 30% H2O2. (A) HSQC showing that the G(8–5)Gglycerol structure was formed from DDC. (B) HSQC NMR spectrum of synthesized S(8–5)Gglycerol. (C) HSQC-TOCSY NMR spectrum of G(8–5)Gglycerol showing that all the red correlations are in the same compound and in the same coupling network, validating the glycerol structure.


11

Supplemental Figure 10. IDDDC Conversion to DDC. The parent phenylcoumaran DDC1 can result from IDDDC12 under radical-generating conditions by a process that is not widely appreciated. We have shown that the benzylic-CH2 compound dihydroconiferyl alcohol, for example, produces, in planta, guaiacylpropan-7,9-diol, via the quinonemethide intermediate followed by water addition, and that the product becomes incorporated into the lignin (Ralph et al., 2009). As we validate here experimentally with a close analog of the primary PCBER product (Supplementary Figure 11), two IDDDC radicals can and do disproportionate to yield one molecule of the original IDDDC and another of its quinonemethide. This quinonemethide is exactly the same intermediate as that produced by 8–5-coupling (dehydrodimerization) of hydroxycinnamyl alcohols and is well-known to rearomatize via internal trapping by the phenolic OH to produce the phenylcoumaran. We contend that this can occur for any of the benzyl-reduced phenylcoumarans, including the PCBER product described in this paper.


12

Supplemental Figure 11. 2D HSQC NMR Spectra of Ag2O Oxidation of TDDC. Ag2O, like peroxidase- H2O2, is a single-electron oxidant (Zanarotti, 1982). (A) The TDDC (as the starting material) contained about 1.6% DDDC after purification. (B) DDDC was significantly increased after a 15-h reaction. (C) DDDC was even more significantly (~ 2.7 times compared to the 15 h reaction) increased after a 36-h reaction.


13

Supplemental Table 1: List of the most differential compounds between PCBER-downregulated and WT plants.

Compo

und ID

Ionis

a-tion

Mod

e

Ratio

PCBER-RNAi/WT

Abundance

(average ion intensity ± SE)

tR

(min)

m/z Chemical

Formula

∆pp

m

MSn Notes

Compounds increasing in PCBER-downregulated plants

M510T613

positi

ve

8055.280

0

WT: NA

PCBER-RNAi-1:

453927 ± 121040

PCBER-RNAi-2:

351601 ± 41204

10.2

1

510.179

2

C24H32O9

N1S1

-

0.059

MS2:167 (20) 205 (25) 209

(100) 235 (49) 312 (14)

330 (31) 353 (18) 357 (40)

371 (62) 389 (91)

Scys(8–O–4)G

MS3(209): 133 (1) 139 (0)

145 (2) 149 (24) 151 (2)

153 (10) 163 (1) 177 (65)

181 (100) 191 (2)

MS3(389): 167 (53) 187

(17) 205 (44) 235 (72) 321

(34) 341 (17) 353 (53) 357

(100) 359 (14) 371 (94)

M718T1009

positi

ve

3787.690

0

WT: NA

PCBER-RNAi-1:

198556 ± 25070

PCBER-RNAi-2:

180213 ± 16370

16.8

2

718.253

0

C35H44O13

N1S1

0.31 MS2:209 (52) 223 (5) 263

(5) 312 (7) 330 (8) 387

(100) 417 (6) 551 (6) 567

(5) 579 (14)

MS3(387): 191 (13) 203 (9)


14

205 (3) 231 (3) 235 (9)

263 (24) 351 (4) 355 (7)

357 (100) 369 (8)

MS3(209): 191 (100)

M540T6

80 positi

ve

3782.690

0

WT: NA

PCBER-RNAi-1:

217449 ± 60184

PCBER-RNAi-2:

160820 ± 22700

11.3

4

540.189

9

C25H34O10

N1S1

0.157 - Scys(8–O–4)S

M510T6

38 positi

ve

2833.740

0

WT: NA

PCBER-RNAi-1:

157381 ± 37063

PCBER-RNAi-2:

125993 ± 18889

10.6

3

510.179

2

C24H32O9

N1S1

-

0.059

MS2: 167 (18) 205 (27)

209 (100) 235 (48) 330

(21) 353 (16) 357 (33) 371

(60) 388 (12) 389 (85)

Scys(8–O–4)G

MS3(209): 133 (1) 145 (4)

149 (28) 151 (2) 153 (12)

163 (0) 167 (1) 177 (63)

181 (100) 191 (2)

MS3(389): 167 (39) 187

(12) 205 (40) 235 (57) 321

(29) 341 (17) 353 (39) 357

(89) 359 (15) 371 (100)

M510T6

85 positi

ve

2519.020

0

WT: NA

PCBER-RNAi-1:

140619 ± 35715

PCBER-RNAI-2:

11.4

1

510.179

2

C24H32O9

N1S1

-

0.059

- Scys(8–O–4)G


15

111283 ± 15146

M702T5

47 positi

ve

2266.450

0

WT: NA

PCBER-RNAi-1:

130356 ± 31704

PCBER-RNAi-2: 96289

± 13355

9.11 702.242

0

C31H44O15

N1S1

-

0.952

MS2: 209 (38) 223 (15)

312 (27) 330 (51) 383 (20)

387 (82) 401 (16) 419 (73)

492 (59) 540 (100)

dilignol +

hexose + cys

MS3(540): 181 (11) 193

(14) 209 (73) 339 (10) 369

(12) 383 (17) 387 (91) 391

(8) 401 (18) 419 (100)

MS3(387): 307 (10) 309 (3)

319 (4) 323 (18) 337 (7)

339 (13) 341 (6) 351 (14)

355 (7) 369 (100)

M540T6

76 positi

ve

1940.890

0

WT: NA

PCBER-RNAi-1: 79781

± 71721

PCBER-RNAi-2:

114308 ± 109639

11.2

7

540.189

9

C25H34O10

N1S1

0.157 MS2: 177 (6) 193 (7) 209

(65) 312 (7) 330 (11) 369

(14) 383 (21) 387 (100)

401 (16) 419 (91)

Scys(8–O–4)S

MS3(387): 291 (4) 307 (9)

309 (4) 319 (4) 323 (17)

337 (5) 339 (12) 351 (16)

357 (5) 369 (100)

MS3(419): 247 (1) 330 (2)

341 (1) 359 (10) 369 (6)

371 (2) 383 (23) 387 (100)


16

391 (8) 401 (17)

M530T6

66 nega

tive

93.9423 WT: 589 ± 245

PCBER-RNAi-1: 60599

± 7646

PCBER-RNAi-2: 48971

± 8281

PCBER-RNAi-4: 56426

± 10234

11.1 530.148

68

C18H24O12

N7

-

0.306

- contains

nitrogen

M671T6

88 nega

tive

16.3310 WT: 8821 ± 1235

PCBER-RNAi-1:

145530 ± 26879

PCBER-RNAi-2:

143236 ± 20950

PCBER-RNAi-4:

143400 ± 20433

11.5 671.203

57

C39H31O9

N2

0.099 MS2: 283 (2) 299 (8) 335

(14) 373 (5) 453 (3) 461

(100) 475 (16) 491 (3) 585

(3) 651 (3)

MS3(461):150 (3) 177 (16)

181 (5) 193 (22) 195 (6)

207 (3) 211 (10) 269 (9)

281 (5) 299 (100)

M717T1

282 nega

tive

15.9942 WT: 5254 ± 1535

PCBER-RNAi-1: 87582

± 14230

PCBER-RNAi-2: 82797

± 21469

PCBER-RNAI-4: 81721

± 21261

21.4 717.225

4

C31H41O19 0.903 -


17

M760T7

85 nega

tive

9.2431 WT: 883 ± 381

PCBER-RNAi-1: 7734 ±

1022


1374


914

13.1 760.281

4

C37H46O16

N1

-

1.062

- contains

nitrogen

M717T5

81 nega

tive

8.5616 WT: 7169 ± 1753

PCBER-RNAi-1: 64785

± 13659

PCBER-RNAi-2: 50952

± 6987

PCBER-RNAi-4: 68398

± 9922

9.7 717.209

34

C27H41O22 -

0.217

-

M1150T

686 nega

tive

7.8974 WT: 809 ± 268


973


1048


1373

11.4 1150.40

218

C42H68O30

N7

0.499 - contains

nitrogen

M682T6

51 nega

tive

7.0920 WT: 2849 ± 807

PCBER-RNAi-1: 22484

± 4265

PCBER-RNAi-2: 19021

± 2392

10.8 682.216

36

C22H40O21

N3

0.560 MS2: 214 (43) 385 (9) 392

(24) 422 (100) 494 (9) 606

(15) 616 (22) 618 (11) 632

(11) 664 (17)

contains

nitrogen


18

PCBER-RNAi-4: 19110

± 4077

M583T5

71 nega

tive

4.4149 WT: 5766 ± 769

PCBER-RNAi-1: 20644

± 5546

PCBER-RNAi-2: 27414

± 5846

PCBER-RNAi-4: 28311

± 6402

9.52 583.202

52

C27H35O14 -

1.217

see Figure 4A in vivo

substrate

M818T8

87 nega

tive

4.1324 WT: 12981 ± 937

PCBER-RNAi-1: 62884

± 10021

PCBER-RNAi-2: 42298

± 4364

PCBER-RNAi-4: 55745

± 7392

14.8 818.207

60

C57H28O4

N3

-

1.136

- contains

nitrogen

M794T4

63 nega

tive

4.1253 WT: 27997 ± 2614

PCBER-RNAi-1:

117048 ± 19852

PCBER-RNAi-2:

105097 ± 12489

PCBER-RNAi-4:

124339 ± 21066

7.72 794.263

91

C36H40O14

N7

0.048 256 (12) 348 (3) 390 (3)

392 (2) 404 (3) 422 (16)

509 (4) 510 (3) 566 (3)

581 (4)

contains

nitrogen

M774T9

41 nega

tive

3.0465 WT: 43708 ± 2799

PCBER-RNAi-1:

146610 ± 22443

15.7 774.217

40

C39H32O11

N7

1.126 MS2:373 (6) 388 (2) 404

(12) 426 (49) 521 (2) 534

(2) 564 (100) 744 (1) 744

contains

nitrogen


19

PCBER-RNAi-2:

116160 ± 14106

PCBER-RNAi-4:

136696 ± 21023

(1) 751 (2)

MS3(564):171 (2) 250 (5)

388 (6) 394 (2) 404 (29)

411 (1) 411 (2) 426 (100)

520 (1) 534 (3)

MS3(426):171 (5) 191 (6)

193 (11) 220 (7) 232 (25)

235 (14) 250 (100) 337 (3)

352 (19) 411 (48)

M980T719

nega

tive

2.6492 WT: 20054 ± 3019

PCBER-RNAi-1: 53286

± 6121

PCBER-RNAi-2: 46377

± 5818

PCBER-RNAi-4: 59718

± 4404

12.0 980.263

43

C38H50O27

N3

-

0.292

- contains

nitrogen

M936T7

50 nega

tive

2.6229 WT: 13173 ± 884

PCBER-RNAi-1: 32897

± 3829

PCBER-RNAi-2: 31611

± 3786

PCBER-RNAi-4: 39148

± 6512

12.5 936.271

09

C49H46O18

N1

-

1.011

MS2:426 (1) 564 (10) 622

(1) 672 (1) 762 (1) 774

(100) 796 (1) 816 (1) 858

(1) 874 (1)

contains

nitrogen


20

MS3(774):250 (1) 356 (1)

373 (13) 404 (11) 411 (4)

426 (49) 534 (4) 546 (2)

564 (100) 598 (9)

Compounds decreasing in PCBER-downregulated plants

M442T60

7 positi

ve

0.0009 WT: 52893 ± 8390

PCBER-RNAi-1: NA

PCBER-RNAi-2: NA

10.1

1

442.207

2

C21H32O9

N1

0.116 MS2:193 (1) 235 (1) 329

(1) 359 (6) 371 (12) 376

(17) 377 (79) 388 (4) 389

(16) 407 (100)

dilignol +

2xwater + NH3

MS3(407): 193 (24) 205 (5)

217 (8) 235 (42) 329 (30)

353 (17) 359 (25) 371

(100) 377 (8) 389 (31)

MS3(377): 167 (2) 177 (29)

191 (2) 193 (39) 205 (75)

309 (3) 327 (2) 341 (16)

345 (2) 359 (100)

M567T13

02 negat

ive

0.0020 WT: 24618 ± 3811

PCBER-RNAi-1: NA

PCBER-RNAi-2: NA

PCBER-RNAi-4: NA

21.7 567.225

22

C19H39O17

N2

-

0.354

- contains

nitrogen

M424T95

2 positi

ve

0.0021 WT: 23755 ± 2292

PCBER-RNAi-1: NA

PCBER-RNAi-2: NA

15.8

6

424.196

6

C21H30O8

N1

0.015 MS2: 193 (11) 209 (14)

317 (7) 357 (31) 371 (18)

379 (7) 383 (15) 389 (100)

dilignol+water+

NH3


21

405 (16) 406 (20)

M423T54

6 negat

ive

0.0110 WT: 5643 ± 913

PCBER-RNAi-1: 55 ±

10

PCBER-RNAi-2: 74 ±

24

PCBER-RNAi-4: 57 ±

19

9.1 423.166

95

C21H27O9 2.11 MS2: 194 (1) 303 (1) 313

(2) 326 (2) 330 (4) 342 (4)

345 (100) 357 (100) 358

(1) 360 (16)

aglycone of in

vivo product

MS3(345): 138 (1) 152 (2)

164 (8) 177 (1) 191 (1)

281 (1) 302 (2) 313 (16)

315 (1) 330 (100)

MS3(357): 177 (1) 179 (2)

295 (1) 299 (1) 310 (1)

314 (3) 325 (2) 327 (1)

329 (1) 342 (100)

M509T525

negat

ive

0.0073 WT: 6896 ± 1604

PCBER-RNAi-1: NA

PCBER-RNAi-2: NA

PCBER-RNAi-4: NA

8.7 509.166

79

C24H29O12 0.669 -

M585T40

0 negat

ive

0.0137 WT: 3658 ± 522

PCBER-RNAi-1: NA

PCBER-RNAi-2: NA

PCBER-RNAi-4: NA

6.68 585.218

14

C27H37O14 -

1.264

see Figure 4B in vivo product

M585T53

3 negat

ive

0.0702 WT: 40719 ± 3500


8.9 585.218

01

C27H37O14 -

1.485

MS2: 197 (6) 327 (3) 375

(8) 389 (3) 405 (23) 423

isomer of in

vivo product


22

1268


2213


1447

(17) 479 (5) 537 (92) 541

(5) 567 (100)

M634T69

1 negat

ive

0.1839 WT: 31803 ± 2276


2796


2225


2281

11.5 634.229

96

C34H36O11

N1

0.908 - contains

nitrogen

M831T14

40 negat

ive

0.5864 WT: 479789 ± 23113

PCBER-RNAi-1:

261444 ± 25081

PCBER-RNAi-2:

261255 ± 15258

PCBER-RNAi-4:

321301 ±27516

24.0 831.287

89

C44H47O16 1.121 MS2: 439 (1) 587 (10) 635

(4) 735 (73) 765 (1) 771

(1) 783 (100) 801 (1) 813

(1) 816 (1)

MS3(783): 350 (0) 380 (0)

409 (0) 557 (0) 587 (10)

735 (100) 751 (0) 753 (0)

765 (0) 768 (5)

MS3(735): 282 (0) 496 (0)

558 (0) 673 (0) 688 (0)

690 (0) 693 (0) 702 (0)


23

705 (2) 720 (100)

M1005T1

432 negat

ive

0.5922 WT: 259234 ± 11337

PCBER-RNAi-1:

142447 ± 11162

PCBER-RNAi-2:

134189 ± 4265

PCBER-RNAi-4:

183917 ± 15993

23.9 1005.38

130

C45H65O25 -

0.736

MS2: 761 (8) 791 (7) 809

(8) 863 (7) 939 (29) 957

(11) 957 (31) 969 (41) 975

(7) 987 (100)

MS3(987): 565 (11) 583

(15) 595 (34) 743 (17) 773

(19) 791 (55) 845 (38) 934

(12) 939 (98) 969 (100)

MS3(969): 373 (23) 563

(17) 595 (100) 725 (16)

743 (28) 773 (24) 791 (32)

921 (31) 939 (19) 951 (65)

M1005T1464

negat

ive

0.5999 WT: 250186 ± 10477

PCBER-RNAi-1:

129522 ± 19187

PCBER-RNAi-2:

142873 ± 4923

PCBER-RNAi-4:

177889 ± 13821

24.4 1005.38

094

C45H65O25 -

1.094

MS2: 617 (11) 761 (10)

809 (13) 939 (38) 957 (13)

957 (52) 969 (28) 969 (21)

975 (12) 987 (100)

An OPLS-DA loading factor of 0.04 and a nested ANOVA significance value of 10-6 were used as threshold. When a peak was below the detection

threshold (NA), a default minimal value of 50 was assigned.


24

Supplemental Table 2: List of compounds detected by GC-MS.

Name m/z tR

(min)

Compound Name Ratio

PCBER-RNAi/WT

Kruskal-

Wallis K Statistic

P

Value*

M371T2522 371 42.04 [1-monohexadecanoylglycerol] 1.34 1.88 0.39

M399T2691 399 44.85 [1-monooctadecanoylglycerol] 1.32 1.65 0.44

M292T1422 292 23.70 [2,3-diOHbutanedioic acid] 0.84 1.88 0.39

M245T1491 245 24.85 [2,4,5-triOHpentanoic acid] 1.88 4.46 0.11

M217T1638 217 27.30 [2-keto-gluconic acid] 0.96 2.19 0.33

M249T2127 249 35.46 [2-O-glycerol-galactoside] 0.55 2.77 0.25

M359T2044 359 34.07 [3-deoxy-arabino-hexaric acid] 1.21 3.04 0.22

M369T2389 369 39.81 [eicosanoic acid] 1.47 1.88 0.39

M254T1345 254 22.41 [erythronic acid] 0.77 0.88 0.64

M66T607 66 10.12 [glycolic acid] 0.86 0.46 0.79

M158T1334 158 22.24 [N-acetylglutamic acid] 0.91 0.46 0.79

M307T1624 307 27.06 [ribonic acid] 0.94 1.28 0.53

M297T2926 297 48.76 4-OH-3-OCH3-phenethylene

glycol

0.22 8.43 0.01

M108T2159 108 35.99 9,12-octadecadienoic acid 2.03 3.30 0.19

M339T2165 339 36.09 9-octadecenoic acid 1.48 3.50 0.17

M103T1509 103 25.16 arabinose 0.94 0.96 0.62

M359T1865 359 31.08 ascorbic acid 1.40 1.88 0.39

M232T1315 232 21.92 aspartic acid 2.42 4.77 0.09

M179T893 179 14.88 benzoic acid 0.93 2.42 0.30

M366T1713 366 28.55 citric acid 1.05 1.65 0.44

M324T1862 324 31.03 coniferyl alcohol 0.66 5.65 0.06

M316T1744 316 29.06 dehydroascorbic acid dimer 1.31 1.42 0.49

M278T1775 278 29.59 fructose 1.02 0.27 0.87

M459T2248 459 37.47 fructose-6-P 0.93 1.50 0.47

M245T1061 245 17.69 fumaric acid 1.17 3.04 0.22

M304T1326 304 22.10 GABA 1.17 1.08 0.58

M204T2842 204 47.37 galactinol 0.90 1.08 0.58

M292T1916 292 31.93 galactonic acid 0.85 1.85 0.40

M494T2648 494 44.13 gentiobiose-like 1.20 0.50 0.78

M204T2670 204 44.50 gentiobiose-like 0.89 2.42 0.30

M189T1931 189 32.18 glucaric acid 0.89 1.65 0.44

M305T1953 305 32.55 glucaric acid 1.08 3.23 0.20


25

M292T1969 292 32.82 glucaric acid-like 0.88 1.19 0.55

M305T1986 305 33.10 glucaric acid-like 1.04 2.58 0.28

M387T2357 387 39.29 gluconic acid-6-P 0.99 0.04 0.98

M191T1905 191 31.76 glucose 0.91 1.88 0.39

M471T2260 471 37.67 glucose-6-P 0.89 0.81 0.67

M156T1455 156 24.25 glutamic acid 1.28 0.27 0.87

M184T1024 184 17.06 glyceric acid 1.10 1.04 0.59

M299T1698 299 28.30 glyceric acid-3-phosphate 0.80 0.13 0.94

M357T1641 357 27.35 glycerol-3-phosphate 1.10 2.00 0.37

M174T982 174 16.37 glycine 0.91 0.27 0.87

M313T1983 313 33.05 hexadecanoic acid 1.34 3.50 0.17

M235T2132 235 35.54 hexose-related 0.75 0.73 0.70

M193T1465 193 24.41 hydroxybenzoic acid 0.57 3.50 0.17

M86T657 86 10.96 hydroxylamine 1.17 1.19 0.55

M433T1937 433 32.28 inositol-like 0.82 1.61 0.45

M245T1716 245 28.60 isocitric acid 1.03 2.19 0.33

M158T964 158 16.06 isoleucine 1.33 1.65 0.44

M288T1397 288 23.28 ketoglutaric acid 0.69 1.09 0.58

M130T575 130 9.59 lactic acid 2.80 1.62 0.44

M265T1277 265 21.28 malate 1.05 0.81 0.67

M245T985 245 16.41 maleic acid 1.09 1.85 0.40

M130T2467 130 41.11 maltose-like 1.17 0.73 0.69

M171T2316 171 38.60 melibiose-like 0.62 0.54 0.76

M129T2336 129 38.93 melibiose-like 1.05 0.81 0.67

M393T2030 393 33.83 myo-inositol 0.95 0.62 0.74

M299T2348 299 39.13 myo-inositol-2-P 1.19 2.35 0.31

M180T971 180 16.18 nicotinic acid 1.06 0.50 0.78

M341T2194 341 36.57 octadecanoic acid 1.38 4.65 0.10

M193T933 193 15.55 phosphate 1.03 1.85 0.40

M156T1316 156 21.94 pyroglutamic acid 1.16 0.27 0.87

M348T1756 348 29.27 quinic acid 1.00 0.81 0.67

M267T2508 267 41.79 salicylic acid glucoside 0.72 3.59 0.17

M116T908 116 15.13 serine 0.86 0.81 0.67

M204T1706 204 28.43 shikimic acid 1.15 2.92 0.23

M247T1003 247 16.71 succinic acid 0.96 1.38 0.50

M452T2563 452 42.72 sucrose 0.84 2.35 0.31

M293T1370 293 22.84 threonic acid 0.64 5.77 0.06


26

M247T1092 247 18.20 threonic acid-1,4-lactone 0.58 4.83 0.09

M117T968 117 16.13 threonine 1.11 2.42 0.30

M361T2377 361 39.62 trehalose-like 0.04 4.36 0.11




M203T2157 203 35.96 Trp 1.18 0.63 0.73

M144T835 144 13.91 valine 1.25 1.50 0.47

M89T1500 89 25.00 xylose 0.09 4.36 0.11

* none of the P values corresponded with a FDR-based Q value lower than 0.05

None of these compounds is significantly differential between PCBER-downregulated plants and WT.


27

Supplemental Table 3: Results of Statistical Analysis Done after Amino Acid Quantification.

AA Kruskal-

Wallis K Statistic

P Value*

Asp 2.868259 0.412386

Glu 6.639173 0.084333

Ser 6.008863 0.11118

Asn 1.854415 0.603166

Gly 5.757397 0.124029

Gln 7.010146 0.071575

His 5.780088 0.122814

Thr 6.549639 0.087725

Arg 5.251935 0.154252

Ala 2.54362 0.46746

Pro 7.495279 0.05768

Tyr 4.87352 0.181296

Cys 6.211281 0.101771

Val 5.000985 0.171725

Met 6.650934 0.083896

Ile 3.469915 0.324685

Leu 3.734027 0.291654

Lys 8.910793 0.030501

Phe 3.7133 0.294133

Trp 7.050246 0.070313

* none of the P values

corresponded with a FDR-

based Q value lower than

0.05

None of the amino acids is significantly differentially abundant between PCBER-downregulated plants

and WT.


28

Supplemental Methods

Sequence of PCBER-RNAi construct

The sequence of the PCBER-RNAi construct is:

5'TCGGAGATGGCAATGCCAAGTTGGTTTTCAATA

AGGAAGATGACATTGGAACCTACACCATCAAAGC

TGTGGATGATGCAAGAACCTTGAACAAGACTGTC

CTAATCAAGCCTCCTAAAAACACCTACTCATTCAA

TGAGCTTGTTGATCTATGGGAGAAAAAGATTGGC

AAAACCCTCGAAAAAACCTTTGTGCCTGAAGAGA

AACTTCTGAAGGACATCCAAGAGTCTCCGATTCC

GATTAATATTGTTCTGTCAATCAACCACTCAGCCC

TCGTTAATGGTGACATGACCAACTTCGAGATTGA

CCCATCATGGGGGCTTGAGGCCTCTGAGCTATAT

CCAGATGTCAAATATACCACTGTGGAAGAGTACC

TTGATCAGTTTGTCTGAGGCACTGGCATCTCCTG

CTCTCC3'

Phenotypic Analysis of Growth

The height of the plants was measured once a week for 15 weeks. Longitudinal regression was applied to

detect possible differences in growth rate between WT and PCBER-downregulated poplars. Statistical

analysis was performed using R vs.2.6.1 (www.r-project.org).

Open-Air Cage-House Trials

Plantlets of the WT, and PCBER-RNAi lines were supplied to Forstbotanik und Baumphysiologie, Georg-

August Universität Göttingen (173 m above sea level, 51° 36' 00" N, 9° 54' 00" E, Germany) in May 2008,

potted and acclimated to outdoor ambient conditions in an open air cage-house. In July 2008, the poplars

(n = 7 per transgenic line, n = 11 for the WT) were planted into boxes (250 cm length x 250 cm

width, 80 cm height) containing compost soil (Vogteier-Erdenwerk, Niederdorla, Germany) in the cage

house. The experimental plants were surrounded by one outer row of border plants. During the growth

seasons the plants were watered regularly. On 18th February 2009 the plants were cut back to a stump of

10 cm height according to coppicing practice. In spring 2009 and 2010 the trees were treated once with

insecticide (TALSTAR, Belchim Crop Protection, Burgdorf, Germany). In 2010, the poplars were fertilized

from June to September once a week with 2 L per plant (2 g L-1HapaphosBlau, Compo GmbH, Münster,

Germany). Plant heights were recorded regularly in 2009 (annual mean temperature 9.3°C) and 2010

(annual mean temperature 8.1°C).

Transmission Electron Microscopy


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29

Small wood sections of poplar twigs were cut approximately 70 cm below the apical bud and immediately

immersed for 4 h in a fixation medium containing 1% (w/v) formaldehyde, 1 mM EGTA, 50 mM cacodylate

buffer, and 5% glutaraldehyde. Subsequently, the tissue was post-fixed with 2% (w/v) osmium tetroxide

overnight at room temperature, stained with 3% (w/v) uranyl acetate in 20% ethanol for 1 h and

dehydrated using a graded series of ethanol. After that, the tissue was embedded in Spurr’s epoxy resin.

Ultrathin sections with a thickness of 70 to 80 nm were cut with a diamond knife on an ultramicrotome

(Reichert-Jung; Ultracut E), transferred onto copper grids coated with formvar and stained with lead

citrate. Sections were observed using a Philips CM 12 transmission electron microscope at 80 kV.

Lignin Quantification

Lignin was quantified by the acetyl bromide method essentially according to Dence, 1992. In brief,

approximately 5 mg of ground xylem was washed with water, ethanol, chloroform, and acetone to prepare

the cell wall residue (CWR). To each sample, 100 μL 25% acetyl bromide in glacial acetic acid was

added followed immediately by 4 µL 60% perchloric acid, the mixture was incubated for 30 min at 70 °C

with shaking. After centrifugation the pellet was washed with 500 μL glacial acetic acid. To the

supernatant, 200 μL of 2 M NaOH and 500 μl glacial acetic acid were added, after which the washings

from the pellet were combined with the supernatant and 700 μL of glacial acetic acid were added. The

solution was shaken and left at room temperature for 20 min. The absorbance at 280 nm was measured

with a NanoDrop spectrophotometer (Thermo Scientific). Lignin concentration was calculated using the

law of Bougeur-Lambert-Beer A = e x l x c [l = 0.1 cm, e = 23.35 L/(g x cm)].

Total Cell Wall and Lignin Analysis by NMR

The whole plant cell wall gel-state NMR samples were prepared as previously described (Kim et al., 2008;

Mansfield et al., 2012). NMR experiments for the whole plant cell wall gel-state samples were performed

as previously described (Kim et al., 2008; Kim and Ralph, 2010). NMR spectra were acquired on a Bruker

Biospin (Billerica, MA) Avance 500 MHz spectrometer equipped with a cryogenically cooled 5-mm triple

resonance (TCI) gradient probe with inverse geometry (proton coils closest to the sample). The central

DMSO solvent peaks were used as internal references (C 39.5, H 2.49 ppm). The 13C–1H correlation

experiment was an adiabatic HSQC experiment (Bruker standard pulse sequence ‘hsqcetgpsisp.2’;

phase-sensitive gradient-edited 2D HSQC using adiabatic pulses for inversion and refocusing) (Kupce

and Freeman, 2007). HSQC experiments were carried out using the following parameters: acquired from

10 to 0 ppm in F2 (1H) with 1000 data points (acquisition time 100 ms), 200 to 0 ppm in F1 (13C) with 400

increments (F1 acquisition time 8 ms) of 80 scans with a 500 msinterscan delay; the d24 delay was set to

0.89 ms (1/8J, J = 145 Hz). The total acquisition time was 5.5 h. Processing used typical matched

Gaussian apodization (GB = 0.001, LB = -0.1) in F2 and squared cosine-bell and one level of linear

prediction (32 coefficients) in F1. Volume integration of contours in HSQC plots used Bruker’s TopSpin

3.1 (Mac version) software; integration was carried out after re-processing without the application of linear

prediction.


30

Dihydroxylation of G(8–5)G (DDC) via H2O2

Enzyme-independent oxidation of cinnamyl alcohol endgroups to their glycerol derivatives was

demonstrated on relevant compounds. DDC (200 mg, 0.56 mmol) was dissolved in acetone (20 mL) and

H2O (2 mL). Hydrogen peroxide (30%, 1.3 mL, 11.2 mmol) was added and the reaction solution stirred for

15 h at room temperature. Solvents were removed directly from the reaction mixture via a rotary

evaporator. A pale yellow oil was obtained in which G(8–5)Gglycerol was produced as a minor, but

significant, product. Analogous treatment of the phenolic acetate derivative of sinapyl alcohol also

produced its glycerol analog.

Ag2O oxidation of TDDC (Tetrahydrodehydrodiconiferyl alcohol)

Conversion of PCBER-type product, α-CH2 opened phenylcoumaran structures, back to the cyclic

phenylcoumarans that are the PCBER substrates, via radical process, was demonstrated on TDDC.

TDDC (50 mg, 0.14 mmol) was dissolved in acetone (10 mL). Ag2O (160 mg, 0.69 mmol) was added, and

the reaction mixture was stirred for 15 h at room temperature. Another similar batch was prepared and

stirred for 36 h under the same conditions. The reaction mixture in each case was filtered through a

sintered glass filter funnel (fine porosity) and the acetone was evaporated off. The crude product was

directly checked by NMR which showed significant conversion to the phenylcoumaran products – See

Supplemental Figure 11 online, and the mechanism for the close analog in Supplemental Figure 10 online.

Metabolite profiling by LC-MS

Metabolites were extracted from 28 WT and 9-10 plants each for PCBER-RNAi-1, PCBER-RNAi-2 and

PCBER-RNAi-4. Approximately 20 mg of frozen ground xylem from each plant was transferred to an

Eppendorf tube, and metabolites were extracted with 500 µL of methanol. The dry weight of the xylem

after methanol extraction was recorded and used for normalization. After removal of the methanol under

reduced pressure, the pellet was dissolved in 200 µL water and 200 µl cyclohexane. The water phase was

collected and 25 µL was analyzed on an Accela UHPLC system (Thermo Electron Corporation, Bremen,

Germany) consisting of an Accela autosampler coupled to an Accela pump and connected to a LTQ FT

Ultra (Thermo-Electron Corporation) MS comprising a linear ion trap (IT) MS connected to a FT-ICR-MS.

The separation was performed on a reversed phase Acquity UPLC BEH® C18 column (150 mm x 2.1 mm,

1.7 μm; Waters, Milford, MA) with aqueous 0.1% acetic acid and acetonitrile/water (99/1, v/v, acidified

with 0.1% acetic acid) as solvents A and B. At a flow of 300 μL/min and a column temperature of 80 °C,

the following gradient was applied: 0 min 5% B, 30 min 45% B, 35 min 100% B. The autosampler

temperature was 5 °C. Samples were analyzed twice, using either positive or negative electrospray

ionization (ESI). For negative ionization, the following parameter values were applied: spray voltage 4.5

kV, capillary temperature 270 °C, sheath gas 40 (arb), aux gas 20 (arb). In positive ionization mode,

these values were 5 kV, 300 °C, 20 (arb) and 53 (arb), respectively. Full FT-ICR-MS spectra between 120

and 1400 m/z were recorded at a resolution of 100,000. For both WT and transgenic lines, one sample


31

(consisting of a pool of extracts from 3 plants) was analyzed using data-dependent MSn. In these cases,

in parallel with the full FT-ICR-MS spectra, three MSn spectra were recorded on the IT-MS using low

resolution data obtained during the first 0.1 s period of the previous full FT-ICR-MS scan: a MS2 scan of

the most abundant m/z ion of the full FT-ICR-MS scan, followed by two MS3 scans of the most abundant

first product ions. MSn scans were obtained with 35% collision energy. The full FT-ICR-MS scans were

converted to netCDF with Xcalibur v. 2.0 SR2 (Thermo-Electron Corporation). Integration and alignment

were performed with the XCMS package (Smith et al., 2006) in R vs. 2.6.1 (www.r-project.org.) using the

following functions: xcmsSet (fwhm = 6, max = 300, snthresh = 2, mzdiff = 0.01), group (bw = 10, max =

300), retcor (method = “loess”, span = 0.2, family = “symmetric”, plottype = “mdevden”). Following

retention time correction, a second peak grouping was performed: group (bw = 8, max = 300). Chemical

formulae of compounds of interest were obtained with the Qual Browser in Xcalibur v 2.0 SR2 and

candidate molecules were searched in the PubChem database

(http://pubchem.ncbi.nlm.nih.gov/search/search.cgi#). Often multiple peaks arise due to in-source

fragmentation. Therefore, peaks that eluted at the same time and for which the abundance correlated

strongly across all chromatograms (Pearson R2 >0.8) were expected to belong to the same compound

and were grouped via an in-house-written R script (Morreel et al., 2014). To reveal significantly different

peak abundances due to PCBER-downregulation, the data were analyzed using both univariate and

multivariate statistics. Using the lm function in R version 2.6.1, nested ANOVA was performed with wild-

type or PCBER-downregulated poplars as main groups and taking the various PCBER lines into account

as subgroups. Principal component analysis (PCA) and orthogonal partial least squares-discriminant

analysis (OPLS-DA) were computed with SIMCA-P11 (www.umetrics.com/simca) following pareto

scaling.

To identify putative substrates and products for PCBER, the profiles were searched for pairs of peaks

differing by a mass corresponding to 2 hydrogen atoms (2.016Da; Morreel et al., 2014), and showing

opposite profiles in the PCBER-downregulated poplars, i.e., the substrate accumulating and the product

decreasing or being absent in the transgenic trees. Furthermore, due to the opening of the

phenylcoumaran ring, the product has one more phenolic hydroxyl group (see Supplemental Figure 6

online); therefore the product should be more hydrophilic and elute earlier than the substrate via reverse

phase chromatography.

Metabolite Profiling by GC-MS

Metabolite extraction and profiling by GC-MS was performed as described previously (Dauwe et al.,

2007). Metabolites were extracted from 4 WT, 4 PCBER-RNAi-1 and 4 PCBER-RNAi-2 plants.

Amino Acid Extraction and Analysis

Amino acids were extracted from homogenized ground xylem tissue using methanol. This extract was

freeze-dried, and re-suspended in a 1:1 mixture of water and cyclohexane; the resulting water phase was

used for analysis on an RP-HPLC column (Waters). Pre-column derivatization with 6-aminoquinolyl-N-


http://www.r-project.org/

http://pubchem.ncbi.nlm.nih.gov/search/search.cgi

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32

hydroxysuccinimidyl carbamate was performed prior to injection, using a commercial labeling kit (Waters

AccQ-Tag), following the manufacturer’s instructions. The detection was performed with UV at 248 nm.

An internal standard of AABA (α-aminobutyric acid) was included for compensation of losses during

preparation and analysis of samples. AABA and all amino acid standards were run at a concentration of

12.5-25 µM.

For the HPLC, buffers A and B comprised sodium acetate (100 mM) at pH 5.50 and pH 8.60,

respectively. Buffer C is acetonitrile (100%). Following sample injection, a pH gradient ran for 65 minutes.

The column was flushed with acetonitrile between each sample.

Purification of Compounds

Young developing xylem was scraped from 28 WT and 32 PCBER-downregulated trees (8 trees from

each of the 4 transgenic lines) from the second harvest, ground in liquid nitrogen and pooled. A total

amount of 100 g of fresh weight was obtained, which was extracted with 600 mL methanol. The filtered

extract was concentrated to 5 mL using a Rotavapor® R II (Buchi) and then a first reversed phase

preparative separation was performed with a RevelerisTM flash chromatography instrument (Grace) using

a Reveleris C18 flash cartridge (12 g; Grace). Solvents A and B were aqueous 0.1% formic acid and

methanol, subjected to vacuum degassing. The following gradient was run using a flow of 40 ml/min: 0

min 5% B, 25 min 50% B, 27 min 100% B. The fixed wavelength UV/Vis absorption detector was set at

270 and 320 nm. Fractions of 20 ml were collected. Their volume was subsequently reduced to

approximately 3 ml with the Buchi rotavapor RII using the same conditions as mentioned above. Further

evaporation to 1 mL was performed with a Savant SC210A Speedvac concentrator (Thermo). The

fractions containing the desired compounds were subjected to two more separations on a Waters 625 LC

system. First a Luna C18 column (10 μm, 10 mm x 250 mm; Phenomenex®) was used with aqueous

0.1% acetic acid and acetonitrile/water (99/1, v/v, acidified with 0.1% acetic acid) as solvents A and B.

The last separation was done on a Platinum™ EPS C18 (10 μm, 10 mm x 250 mm) column with aqueous

solvent adjusted to neutral pH with 0.1% triethylammonium acetate buffer and 0.05% trimethylamine and

acetonitrile/water (99/1, v/v) as solvents A and B. After each separation, fractions were checked for the

presence of the compounds of interest using an Acquity™ Ultra Performance LC system connected to a

LTQ XL Ultra (Thermo-Electron Corporation) linear ion trap MS. The separation was performed on a

reversed phase Acquity UPLC® BEH C18 column (150 mm x 2.1 mm, 1.7 μm; Waters, Milford, MA) with

aqueous 0.1% formic acid and acetonitrile/water (99/1, v/v, acidified with 0.1% formic acid) as solvents A

and B. Separation conditions were the same as for metabolite profiling. It is important to mention that

although the purification was focused on the glycosylated product (see results sections on MS and NMR

analysis of the PCBER substrate and product) at the end of the purification a mixture of glycosylated

product and aglycone was obtained, indicating that the glycosylated product is not very stable and was

therefore present in low amounts in the fraction analyzed by NMR.


33

Compound 26 Hex-G(8–O–4)G (see Supplementary Figure 6), was also isolated in one of the fractions

used to purify the PCBER substrate. It was identified by MSn due to a neutral loss corresponding to a

hexose (-162 Da) and a further fragmeyntation pattern consistent with the one previously described for

G(8–O–4)G (Morreel et al., 2010a).

Chemical Synthesis

Preparation of compound 1; G(8–5)G (DDC), compound 3, G(8–5)Gglycerol, compound 5; S(8–5)G,

compound 7; G(8–5)FA, compound 9; H(8–5)H, compound 11; G(8–5)DHCA (DDDC), compound 21;

S(8–8)S, compound 23; G(8–8)G, and compound 24; H(8–8)H were previously described (Morreel et al.,

2004; Morreel, et al., 2010; Supporting Information).

Compound 12, reduced G(8–5)DHCA, 2-(1-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propan-2-yl)-4-(3-

hydroxypropyl)-6-methoxyphenol (TDDDC) was prepared in the same conditions as compound 11

(Morreel, et al., 2010a; Supporting Information), but a longer reaction time (15 h) was needed to complete

the reaction.

Compound 13, S(8–5)FA-Et, (E)-ethyl 3-(2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(hydroxymethyl)-7-

methoxy-2,3-dihydrobenzofuran-5-yl)acrylate, was synthesized with horseradish peroxidase. 1H-NMR (d6-

acetone, 500 MHz): 1.26 (3H, t, J = 7.1 Hz, CH3), 3.61 (1H, m, Sβ), 3.80 (6H, s, S-OMe), 3.90 (2H, m,

Sγ), 3.91 (3H, s, F-OMe), 4.18 (2H, q, J = 7.1 Hz, CH2), 5.61 (1H, d, J=7.0 Hz, Sα), 6.39 (1H, d, J=15.9

Hz, F8), 6.75 (2H, s, S2,6), 7.24 (1H, m, F2), 7.25 (1H, m, F6), 7.60 (1H, d, J = 15.9 Hz, F7). 13C NMR:

14.34 (CH3), 54.36 (Sβ), 56.24 (F-OMe), 56.40 (S-OMe), 60.36 (CH2), 64.18 (Sγ), 89.38 (Sα), 104.54

(S2,6), 113.12 (F2), 116.01 (F8), 118.76 (F6), 129.00 (F1), 130.95 (F5), 132.72 (S1), 145.49 (F7), 145.60

(F3), 148.73 (S3,5), 148.98 (S4), 151.56 (F4), 167.36 (F9).

Compound 15, Et-FA(8–5)G, (E)-ethyl 2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxyprop-1-en-1-yl)-7-

methoxy-2,3-dihydrobenzofuran-3-carboxylate, was prepared with horseradish peroxidase as described

previously (Zhang et al., 2009).

Compound 17, G(8–5)G(5–5)G(8–5)G, was prepared as a mixture of two closely related isomers by

radical coupling of DDC 1 [G(8–5)G] under conditions described in previous publication. 16.0 % Yield.1H-

NMR (d6-acetone, 500 MHz): 3.58 (m. 1H, Aβ), 3.82 (B-OMe), 3.83 (A-OMe), 3.82-3.88 (m, 2H, Aγ), 4.18

(br-s, 2H, Bγ), 5.59 (d, J = 5.9 Hz, α-H), 6.22 (br-d, J =15.9 Hz, 1H, Bβ), 6.50 (br-d, J =15.9 Hz, 1H,Bα),

6.91 (br-s, 1H, B2), 6.94 (br-s, 1H, A6), 6.97 (br-s, 1H, B6), 7.03 (br-s, 1H, A6). 13C NMR: 54.63/54.67 (A

β), 56.29 (B-OMe), 56.38 (A-OMe), 63.34 (Bγ), 64.50 (Aγ), 88.47 (Aα), 109.14 (A2), 111.54/111.57 (B2),

116.0 (B6), 121.73/121.76 (A6), 125.99 (A5), 128.22 (Bβ), 130.32/130.36 (B5), 130.5 (Bα), 131.81 (B1),

133.54/133.58 (A1), 144.44/144.46 (A4), 145.07 (B3), 148.64 (A3), 148.82 (B4).

Compound 19, G(8–5)G(4–O–5)G(8–5)G, was produced as a mixture of the two closely related isomers

in low (3.6%) yield from the same coupling reaction as compound 17 above. 1H-NMR (d6-acetone, 500


34

MHz): 3.45 (m. 1H, Aβ), 3.53 (m, 1H, Bβ), 3.74 (B-OMe), 3.84 (A-OMe), 3.86 (C/D-OMe), 3.75-3.90 (m,

4H, Aγ/Bγ), 4.18 (br-s, 4H, Cγ/Dγ), 5.55 (d, J =6.0 Hz, 1H, Aα), 5.62 (d, J =5.9 Hz, 1H, Bα), 6.23 (m, 2H,

Cβ/Dβ), 6.52-6.49 (m, 2H, Cα/Dα), 6.53 (A6), 6.85 (A2), 6.91 (B6), 6.93 (B5), 6.95 (C2/D2), 6.97 (C6/D6),

7.11 (B2). 13C NMR: 54.86/54.88 (Bβ), 54.96/54.98 (Aβ), 56.15, 56.31, 56.31, 56.57 (OMes), 63.33 (Cγ),

63.33 (Dγ), 64.56/64.63 (Aγ/Bγ), 88.04 (Aα), 88.07 (Bα), 105.63/105.68 (A2), 111.42 (B2), 111.56/111.73

(C2/D2), 116.01/116.02 (C6/D6), 116.02 (B5), 118.80/118.82 (B6), 128.33/128.43 (Cα/Dα),

130.01/130.03 (B1), 130.40/130.42 (Cβ/Dβ), 131.94/131.06 (C1/D1), 133.73 (A1), 138.13/138.16 (A4),

145.06 (A5), 145.16 (C3/D3), 146.48/146.51 (B4), 148.54/148.74 (C5/D5), 148.76 (C4/D4), 149.69 (A3),

151.4 (B3).

Compound 25, G(8–O–4)G, 1-(4-hydroxy-3-methoxy-phenyl)-2-[4-(3-hydroxy-propenyl)-2-methoxy-

phenoxy]-propane-1,3-diol, (8–O–4)-dehydrodiconiferyl alcohol, was prepared using the Cu(OAc)2 system

(Landucci et al., 1995).

Compound 27, S(8–O–4)G(8–5)G, 1-(4-hydroxy-3,5-dimethoxy-phenyl)-2-{4-[3-hydroxy-methyl-5-(3-

hydroxy-propenyl)-7-methoxy-2,3-dihydro-benzofuran-2-yl]-2-methoxy-phenoxy}-propane-1,3-diol, was

prepared via traditional synthetic β-ether lignin model methods as previously described (Morreel et al.,

2004).

Compound 28, Glc-G(8–5)G, (E)-2-(hydroxymethyl)-6-(4-(3-(hydroxymethyl)-5-(3-hydroxyprop-1-en-1-yl)-

7-methoxy-2,3-dihydrobenzofuran-2-yl)-2-methoxyphenoxy)tetrahydro-2H-pyran-3,4,5-triol, was

synthesized as described previously (Teutonico et al., 1991).

Compound 29, Me-pCA(8–5)Me-pCA, (E)-methyl 2-(4-hydroxyphenyl)-5-(3-methoxy-3-oxoprop-1-en-1-

yl)-2,3-dihydrobenzofuran-3-carboxylate, was prepared with horseradish peroxidase as described

previously (Yoshihara et al., 1983).

Compound 30, C(8–O–4)C, (E)-4-(3-(hydroxymethyl)-7-(3-hydroxyprop-1-en-1-yl)-2,3-dihydro-

benzo[b][1,4]dioxin-2-yl)benzene-1,2-diol, was prepared with horseradish peroxidase as described

previously (Matsumoto et al., 1999).

Compound 31, G(8–O–4)CA-Me, (E)-methyl 3-(3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-

dihydrobenzo[b][1,4]dioxin-6-yl)acrylate, was prepared by a cross-coupling reaction between coniferyl

alcohol and methyl caffeate as described previously (She et al., 1998).

Compound 32, S(8–5)Gglycerol, 1-(2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-

dihydrobenzofuran-5-yl)propane-1,2,3-triol, was prepared using compound 5, S(8–5)G, through

Sharpless catalytic asymmetric dihydroxylation reactions using AD-mix-α, (DellaGreca et al., 1998; Yue et

al., 2012). 1H-NMR (D2O, 500 MHz): δ 3.23 (1H, m, Bγ1), 3.32 (1H, m, Bγ2), 3.51 (1H, m, Aβ), 3.52 (3H,

s, A-OMe), 3.67 (1H, m, Bβ), 3.71 (3H, s, B-OMe), 3.74 (2H, m, Aγ), 4.45 (1H, d, J=6.6 Hz, Bα), 5.44 (1H,

d, J=6.2 Hz, Aα), 6.58 (2H, br s, A2,6), 6.79 (1H, br s, B6), 6.82 (1H, br s, B2). 13C NMR (D2O, 125 MHz):


35

δ 52.62 (Aβ), 55.85 (A-OMe& B-OMe), 62.37 (Bγ1 & Bγ2), 62.51 (Aγ), 74.00 (Bα), 75.45 (Bβ), 88.14 (Aα),

103.35 (A2,6), 110.87 (B2), 115.47 (B6).

Compound 33, reduced S(8–5)Gglycerol, 1-(4-hydroxy-3-(1-hydroxy-3-(4-hydroxy-3,5-

dimethoxyphenyl)propan-2-yl)-5-methoxyphenyl)propane-1,2,3-triol, was obtained by reduction of the

compound 32 using Pd/C (10% Pd). 1H-NMR (D2O, 500 MHz): δ 2.65 (1H, m, Cα1), 2.84 (1H, m, Cα2),

2.89 (1H, m, Dγ1), 3.04 (1H, m, Dγ2), 3.31 (1H, m, Cβ), 3.49 (1H, m, Dβ), 3.52 (3H, s, C-OMe), 3.64 (3H,

s, D-OMe), 3.72 (1H, m, Cγ), 4.26 (1H, m, Dα), 6.19 (2H, br s, C2,6), 6.48 (1H, br s, D6), 6.66 (1H, br s,

D2). 13C NMR (D2O, 125 MHz): δ 36.17 (Cα1 & Cα2), 43.51 (Cβ), 56.00 (C-OMe& D-OMe), 62.09 (Dγ1 &

Dγ2), 64.48 (Cγ), 74.22 (Dα), 75.43 (Dβ), 106.38 (C2,6), 108.38 (D2), 119.34 (D6).


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Supplemental References:

Dauwe, R., Morreel, K., Goeminne, G., Gielen, B., Rohde, A., Van Beeumen, J., Ralph, J., Boudet, A.M., Kopka, J., Rochange, S.F., Halpin, C., Messens, E., and Boerjan, W. (2007). Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration. Plant J. 52: 263-285.

DellaGreca, M., Fiorentin, A., Monaco, P., and Previtera, L. (1998). Enantioselective synthesis of phenylpropanetriols. Synth. Commun. 28: 3693–3700.

Dence, C.W. (1992). Methods in Lignin Chemistry (Springer Series in Wood Science) (Springer).

Gang, D.R., Kasahara, H., Xia, Z.Q., Vander Mijnsbrugge, K., Bauw, G., Boerjan, W., Van Montagu, M., Davin, L.B., and Lewis, N.G. (1999). Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases. J. Biol. Chem. 274: 7516–27.

Kim, H. and Ralph, J. (2010). Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Org. Biomol. Chem. 8: 576–91.

Kim, H., Ralph, J., and Akiyama, T. (2008). Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6. BioEnergy Res. 1: 56–66.

Kupce, E. and Freeman, R. (2007). Compensated adiabatic inversion pulses: broadband INEPT and HSQC. J. Magn. Reson. 187: 258–65.

Landucci, L.L., Luque, S., and Ralph, S. (1995). Reaction of p-hydroxycinnamyl alcohols with transition metal salts. II: Preparation of guaiacyl/syringyl di-, tri-, and tetralignols. J. Wood Chem. Technol. 15: 493–513.

Mansfield, S.D., Kim, H., Lu, F., and Ralph, J. (2012). Whole plant cell wall characterization using solution-state 2D NMR. Nat. Protoc. 7: 1579–89.

Matsumoto, K., Takahashi, H., Miyake, Y., and Fukuyama, Y. (1999). Convenient syntheses of neurotrophic americanol A and isoamericanol A by HRP catalyzed oxidative coupling of caffeic acid. Tetrahedron Lett. 40: 3185–3186.

Morreel, K., Ralph, J., Kim, H., Lu, F., Goeminne, G., Ralph, S., Messens, E., and Boerjan, W. (2004). Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem. Plant Physiol. 136: 3537–3549.

Morreel, K., Kim, H., Lu, F., Dima, O., Akiyama, T., Vanholme, R., Niculaes, C., Goeminne, G., Inzé, D., Messens, E., Ralph, J., and Boerjan, W. (2010). Mass spectrometry-based fragmentation as an identification tool in lignomics. Anal. Chem. 82: 8095–8105.

Morreel, K., Saeys, Y., Dima, O., Lu, F.C., Van de Peer, Y., Vanholme, R., Ralph, J., Vanholme, B., and Boerjan, W. (2014). Systematic structural characterization of metabolites in Arabidopsis via Candidate Substrate-Product Pair networks. Plant Cell 26: 929-945.

Ralph, J., Schatz, P.F., Lu, F., Kim, H., Akiyama, T., and Nelsen, S.F. (2009). Quinone methides in lignification. In Quinone Methides, S. Rokita, ed (Wiley-Blackwell: Hoboken: NJ), pp. 385–420.


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She, X., Qi, S., Gu, W., and Pan, X. (1998). Study on the total synthesis of neolignans(I): Synthesis of the methyl ethers of americanin A and Americanol A. J. Chem. Res.: 436–437.

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Zanarotti, A. (1982). Preparation and reactivity of 2,6-dimethoxy-4-allylidene-2,5-cyclohexadien-1-one (vinyl quinone methide) a novel synthesis of sinapyl alcohol. Tetrahedron Lett. 23: 3815–3818.

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