Post on 08-Feb-2020
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
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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Supplemental Figure 3. Cellulose quantification. Cellulose quantification showing no significant difference between WT and PCBER-downregulated poplars (Data show means ± SD , n = 5).
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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)
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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)
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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 -
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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M760T7
85 nega
tive
9.2431 WT: 883 ± 381
PCBER-RNAi-1: 7734 ±
1022
PCBER-RNAi-2: 7703 ±
1374
PCBER-RNAi-4: 9048 ±
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
PCBER-RNAi-1: 7080 ±
973
PCBER-RNAi-2: 6051 ±
1048
PCBER-RNAi-4: 6036 ±
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
PCBER-RNAi-1: 1898 ±
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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1268
PCBER-RNAi-2: 4464 ±
2213
PCBER-RNAi-4: 2218 ±
1447
(17) 479 (5) 537 (92) 541
(5) 567 (100)
M634T69
1 negat
ive
0.1839 WT: 31803 ± 2276
PCBER-RNAi-1: 6753 ±
2796
PCBER-RNAi-2: 4126 ±
2225
PCBER-RNAi-4: 6670 ±
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)
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
M362T2457 362 40.94 trehalose-like 1.10 7.65 0.02
M271T2662 271 44.37 trehalose-like 0.74 3.28 0.19
M361T2751 361 45.85 trehalose-like 0.55 2.93 0.23
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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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-
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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.
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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):
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
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).
Supplemental Data. Niculaes et al. (2014). Plant Cell 10.1105/tpc.114.125260
36
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