Document S1. Supplemental Experimental Procedures, Figures S1 ...
Fig. S1 Figure S1. Multiple alignment of the selected OSC sequences from Barbarea vulgaris and...
21
Fig. S1 Figure S1. Multiple alignment of the selected OSC sequences from Barbarea vulgaris and Arabidopsis thaliana, using the software Muscle; this alignment was used to construct the phylogenetic tree in Fig 2.
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Bv1460 Bv14724 Bv14843 Bv Bv1520 Bv6614 Bv4315 Bv1621 Bv2426 Bv Mb chromosome 4 A. thaliana Bv1540 Bv4110 Bv15515 p10m p11m Bv15629 p40m p41m p41m p41m p15m p15m p15m CYP716A 0 Bv15918 Bv Bv1650 Bv16714 Bv Mb 14.2 chromosome 5 A. thaliana CYP72A8 Bv70Bv740 Bv5611 Bv3213 Bv7818 Bv8321 Bv2924 Bv Bv1733 Bv12442 Bv Bv1330 Bv2012 p35m p14m Bv11327 p14m p41m Bv10752 p41m Bv1100 p10m p40m p40m Mb 4.9 chromosome 3 A. thaliana AB CD dominance p41m Bv1239 p38m p35m p14m p16m Bv6533 Bv3640 p14m p14m Bv15 52 Bv11658 p41m p38m p40m p40m p41m Bv12114 p10m Bv3528 Bv Bv120 Bv Bv Bv12835 Bv Bv8650 Bv LUP5 0 Bv Bv9633 Bv Bv600 Bv63-15 p40m p11m LUP2 17 p40m p16m p38m Mb chromosome 1 A. thaliana p10m Bv p15m p14m Bv1370 Ra1229 Bv13936 UGT73C 45 Bv14550 Bv14354 Bv14161 Bv Mb 7.1 chromosome 2 A. thaliana Bv Bv Bv136 2S dominance A BCD 2R dominance QTL for P-type saponins QTL for glucosinolates QTL for G-type saponins A. Hederagenin cellobioside B. Oleanolic acid cellobioside C. Gypsogenin cellobioside D. 4-Epihederagenin cellobioside 1-4 QTL for flea beetle resistance Fig. S2 Figure S2. B. vulgaris QTL map showing 17 linkage groups by vertical bars, aligned to the five A. thaliana chromosomes. QTLs for flea beetle resistance, saponins of the G- and P-type, and glucosinolates are indicated by vertical colored bars, and positions of the six mapped genes for saponin biosynthesis by circles. Genetic distances (cM) are listed at the left of each linkage group and genetic markers (AFLP, SSR) at the right; corresponding marker positions on the A. thaliana chromosomes are indicated in millions of base pairs (Mb). QTLs for the four unknown saponins from the P-type are marked 1 to 4; see Kuzina et al. (2011) for more details; two of these were dominant, as were two QTLs for glucosinolates. Confidence intervals, explained variation and position of the maximal LOD score are in Table S2. AFLP markers are designated with the MseI (m) and PstI (p) primer combinations followed by molecular weight (bp).
Transcript of Fig. S1 Figure S1. Multiple alignment of the selected OSC sequences from Barbarea vulgaris and...
Fig. S1
Figure S1. Multiple alignment of the selected OSC sequences from Barbarea vulgaris and Arabidopsis thaliana, using the software Muscle; this alignment was used to construct the phylogenetic tree in Fig 2.
Fig. S1, continued
Bv1460
Bv14724
Bv14843
Bv1869
13
Bv1520
Bv6614Bv4315Bv1621Bv2426
Bv15334
14
1.4
0.1
15.0
15.8
12.8
17.016.9
16.5
5.8
Mb
chro
mos
ome
4A
. tha
liana
Bv1540
Bv4110Bv15515p10m50-43318p11m48-8124Bv15629
p40m49-22039
p41m49-30258
p41m60-49266
p41m60-13085
p15m60-7597p15m60-76101p15m60-292107
15
CYP716A0
Bv15918
Bv16126
16
Bv1650
Bv16714
Bv626
1723.4
26.4
3.5
6.4
3.54.0
16.316.5
Mb
14.2
chro
mos
ome
5A
. tha
liana
CYP72A8
Bv70 Bv740
Bv5611Bv3213Bv7818Bv8321Bv2924Bv3426
28
Bv1733Bv12442
Bv6453
10
Bv1330
Bv2012
p35m62-10919p14m59-24425Bv11327p14m59-37235p41m60-12639
Bv10752
p41m49-11566
11
Bv1100
p10m50-1078
p40m49-46617
p40m49-47529
12
9.8
15.5
23.4
1.31.30.81.41.83.52.84.74.45.3
Mb
4.9
chro
mos
ome
3A
. tha
liana
A BC D
12
3 4
dom
inan
ce
p41m60-2150
Bv1239
p38m49-13521p35m62-13926p14m48-22729p16m49-34132Bv6533Bv3640p14m59-38343p14m48-39450Bv1552Bv11658p41m60-19464
570
p38m49-27589
p40m49-178114
1
p40m49-1370p41m49-1382
Bv12114p10m50-11720
Bv3528
Bv12635
2
Bv120
Bv51-212
Bv87-221
Bv12835
Bv87-148Bv8650
Bv89-164
3
LUP50
Bv89-214
Bv9633
Bv3147
4
5Bv600Bv63-15p40m49-26611p11m59-25112
LUP217
p40m49-26527
p16m49-13834
p38m49-19143
9.07.54.84.1
0.6
18.920.720.122.922.823.923.323.922.825.1
25.0
25.1
25.4
29.7
Mb
28.3
25.7
chro
mos
ome
1A
. tha
liana
p10m50-4210
Bv13521
6
p15m60-1330
1220
p14m47-20140
7
8
Bv1370
Ra1229
Bv13936
UGT73C45Bv14550Bv14354
Bv14161Bv2365
9
7.8
16.115.3
19.618.2
15.4
12.0
13.5
9.5
9.8
Mb
7.1
chro
mos
ome
2A
. tha
liana
Bv270
20
31
Bv51-142
Bv136
2S
dom
inan
ce
A B C D2R
dom
inan
ce
QTL for P-type saponinsQTL for glucosinolates
QTL for G-type saponinsA. Hederagenin cellobiosideB. Oleanolic acid cellobiosideC. Gypsogenin cellobiosideD. 4-Epihederagenin cellobioside
1-4QTL for flea beetle resistance
Fig. S2
Figure S2. B. vulgaris QTL map showing 17 linkage groups by vertical bars, aligned to the five A. thaliana chromosomes. QTLs for flea beetle resistance, saponins of the G- and P-type, and glucosinolates are indicated by vertical colored bars, and positions of the six mapped genes for saponin biosynthesis by circles. Genetic distances (cM) are listed at the left of each linkage group and genetic markers (AFLP, SSR) at the right; corresponding marker positions on the A. thaliana chromosomes are indicated in millions of base pairs (Mb). QTLs for the four unknown saponins from the P-type are marked 1 to 4; see Kuzina et al. (2011) for more details; two of these were dominant, as were two QTLs for glucosinolates. Confidence intervals, explained variation and position of the maximal LOD score are in Table S2. AFLP markers are designated with the MseI (m) and PstI (p) primer combinations followed by molecular weight (bp).
55.1
73.1
95.1129.1
147.1175.1
189.2
203.2
218.2
257.2279.2 408.4 498.50
20
40
60
80
100[%]
100 200 300 400 500 600 700m/z
Rel
ativ
e A
bund
ance
RT: 22.50 min
55.1
75.1
95.1129.1
147.1
175.1
189.2
218.2
231.2 279.2 361.2408.4 498.50
20
40
60
80
100
[%]
100 200 300 400 500 600 700m/z
Rel
ativ
e A
bund
ance
RT: 22.69 min
a bβ-amyrin α-amyrin
Fig. S3
Figure S3. EI-MS fragmentation patterns and retention times (RT) of trimethylsilyl (TMS) derivatives of 13 authentic triterpene standards and five tentatively identified triterpenoids (unknowns 1-5). Tentatively identified triterpenoids correspond to peak numbers as illustrated in Fig. 3. EI-MS patterns, RT and structures of (a) β-amyrin, (b) α-amyrin, (c) lupeol, (d) erithrodiol, (e) uvaol, (f) betulin, (g) oleanolic acid, (h) oleanolic aldehyde, (i) betulinic acid, (j) betulinic aldehyde, (k) ursolic acid, (l) ursolic aldehyde, (m) hederagenin were compared against authentic standards. Tentative identification of triterpenoids (unknowns 1-5 (n-u)) were based on common EI-MS fragmentation patterns observed for most trimethylsilylated triterpenes and mass spectral comparison with authentic standards. Characteristic fragmentation patterns represented by loss of methyl (-15 m/z), followed by loos of dimethylsilyl oxonium (-75 m/z) and loss of the second methyl (-15 m/z) group from the final trimethylsilylated products (v) and allowed estimation of molecular masses of unknowns 1-5 (Table S8).
55.1
75.1
95.1109.1
121.1135.2
175.2
189.2
203.2
218.2
231.2
245.2257.2
279.2299.3325.3
369.4393.4
483.4
498.5
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700m/z
Rel
ativ
e A
bund
ance
RT: 22.73 min
55.1
75.1
91.1
147.1
216.2
281.1 361.1
496.4
567.5
RT: 23.3 min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
erythrodiol
55.1
75.1
91.1
147.1
203.2
281.1361.1
429.2
496.5
567.5
RT: 23.5min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
uvaol
55.1
73.1
91.1
147.1
204.1
281.0361.2
393.3 483.4
567.5
RT: 23.6 min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
betulin
c dlupeol
m/z
m/z m/z
e f
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
Fig. S3, continued
55.1
73.1
105.1129.1
189.2
203.2
219.2241.2279.2306.2
320.2
393.4
482.4
585.50
20
40
60
80
100
[%]
100 200 300 400 500 600 700
RT: 23.76 min
75.1
105.1129.1
203.2
279.2
RT: 23.8min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
oleanolic aldehyde
H
73.1
95.1
129.1
189.2
257.2
292.2
353.3393.4 483.4 585.5
RT: 23.9 min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
betulinic acid
55.1
75.1
91.1129.1
189.2
279.2 383.3
484.5512.5
RT: 23.9min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
betulinic aldehyde
H
Rel
ativ
e A
bund
ance
oleanolic acid
m/zm/z
m/z m/z
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
g h
i j
Fig. S3, continued
55.1
73.1
133.1
203.2
256.2279.3
320.2
393.4482.5
585.5
RT: 24.0 min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
55.1
75.1
91.1
133.1
203.2
232.2279.3
RT: 24.1 min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
H
ursolic aldehyde
55.1
73.1
105.1
147.1
203.2
278.2
320.2
391.3570.5
24.5 min
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700
hederagenin
ursolic acid
m/z m/z
m/z
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
k l
m
Fig. S3, continued
55.1
75.1
129.1
147.1
159.1
190.2
216.2
243.1
279.2
291.2
306.2
331.1
361.2
406.3496.4
586.60
20
40
60
80
100[%]
100 200 300 400 500 600 700m/z
Rel
ativ
e A
bund
ance
RT: 23.09 min
55.1
73.1
91.1103.1
129.1
147.1
201.2
217.1243.2255.2
271.1291.2331.2
361.2379.3
451.4
494.5505.4
541.5584.5
0
20
40
60
80
100[%]
100 200 300 400 500 600 700m/z
Rel
ativ
e A
bund
ance
RT: 23.67 min
55.1
73.1
103.1
129.1
147.1
189.2
201.2
214.2
253.2291.2 361.1389.4
481.4
494.4
584.5
0
20
40
60
80
100
[%]
100 200 300 400 500 600m/z
Rel
ativ
e A
bund
ance
RT: 23.15 min RT: 23.70 min73.0
103.0
129.0201.1
253.1291.2 389.3
494.4
584.5
0
20
40
60
80
100[%]
100 200 300 400 500m/z
Rel
ativ
e A
bund
ance
600
unknown-1 unknown-4
unknown-2unknown-2 identified in the GC-MS analysis of
saponified leaf extracts of the G-type B. vulgaris
n o
p q
Fig. S3, continued,
55.1
73.1
103.1
129.1
147.1
189.1
201.2
214.2
253.2 304.2361.2389.3
481.4494.4
584.5
0
20
40
60
80
100[%]
100 200 300 400 500 600m/z
Rel
ativ
e A
bund
ance
RT: 23.29 min RT: 23.90 min73.0
103.0
129.0201.1
253.1 304.2389.3
494.4
584.5
0
20
40
60
80
100[%]
100 200 300 400 500m/z
Rel
ativ
e A
bund
ance
600
481.4
55.1
73.1
91.1
129.1
147.1
171.1
187.2
238.1255.1271.1
304.2
347.2361.2391.4413.4
481.4
508.4 583.4
598.5
0
20
40
60
80
100
[%]
100 200 300 400 500 600m/z
Rel
ativ
e A
bund
ance
RT: 24.20 min RT: 24.90 min73.0
95.0
129.0147.0
187.1
238.1 304.1 368.2413.2
481.4
508.4 557.1
598.4
0
20
40
60
80
100[%]
100 200 300 400 500m/z
Rel
ativ
e A
bund
ance
600
unknown-3
unknown-5
unknown-3 identified in the GC-MS analysis of saponified leaf extracts of the G-type B. vulgaris
unknown-5 identified in the GC-MS analysis of saponified leaf extracts of the G-type B. vulgaris
r s
t u
Fig. S3, continued
CH3-
- 15 m/zOSi
CH3
H3C
CH3
499 m/zOSi
H3C
H3C
..+
484 m/z
Si
CH3
CH3
HO+
- 75 m/z
C .+409 m/z
CH3
- 15 m/zHC
+394 m/z
-
360 380 400 420 440 460 480 5000
1
2
3499.0
393.9
408.9
483.9365.6
377.3 455.5425.4 469.6441.3
m/z
[%]
55.1
75.1
95.1
147.1
218.2
281.1 361.2499.0
0
20
40
60
80
100
[%]
100 200 300 400 500 600 700m/z
Zoomed in
vCharacteristic EI-MS of β-amyrin-tms
Rel
ativ
e A
bund
ance
Fig. S3, continued
731.5 824.2 1124.11428.0
1268.6
-MS, 14.3min #974
619.8 781.9944.0
1281.8
1106.1
-MS2(1268.6), 14.3min #975
457.5
619.5781.6
943.6
-MS3(1269.0->1106.7), 14.3min #976
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
739.1 824.4 966.4 1107.31282.2
1428.2
1429.1
-MS, 14.1min #978
455.6617.7
821.71145.7
1267.9
779.8
-MS2(1429.1), 14.1min #979
455.5
617.5
-MS3(1428.8->780.6), 14.2min #980
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
1282.7
-MS, 14.3min #990
795.9
-MS2(1282.7), 14.3min #991
407.3471.4
633.5
-MS3(1283.0->796.7), 14.3min #992
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
459.7 730.9 854.1 981.1 1122.4 1428.0
1268.1
-MS, 13.8min #938
619.7 781.8
943.9
1106.1
-MS2(1268.1), 13.8min #939
457.5
619.5 781.7
943.7
-MS3(1268.6->1106.6), 13.9min #940
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 1 Peak 2
Peak 3 Peak 4
Fig. S4 (a)
Figure S4. LC-MS/MS spectra of tentatively identified saponins produced by expressing (a) LUP5G + CYP716A80 + UGT73C11 and (b) LUP2P + CYP716A80 + UGT73C11 in N. benthamiana, as illustrated in Fig. 4 left (LUP5G) and right (LUP2P) panels, respectively. Mass spectra numbers correspond to peak numbers in Fig. 5. Mass spectra were recorded in negative mode, thus proposed aglycone masses correspond to [M – 1] -.
756.1 843.1 946.8 1152.6
1430.0
1266.3
-MS, 14.8min #1005
781.6 869.6
983.6
1105.71220.0
617.6
-MS2(1266.3), 14.8min #1006
438.5
-MS3(1266.8->618.1), 14.8min #1007
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
814.3 983.8 1268.4 1513.9
1428.3
-MS, 14.9min #1018
617.7
1145.9
1265.7
779.9
-MS2(1428.3), 15.0min #1019
455.5
617.5
-MS3(1428.8->780.0), 15.0min #1020
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
867.2 964.2 1513.8
1267.9
-MS, 15.2min #1035
617.9
780.0
943.8 1105.9
1270.8
780.8
-MS2(1267.9), 15.2min #1036
455.4
617.5
-MS3(1268.3->779.1), 15.2min #1037
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
418.4 536.6 641.3 837.7 963.71419.9
1515.9
1267.3
-MS, 15.5min #1054
617.7
779.7
943.7 1145.7780.6
-MS2(1267.3), 15.5min #1055
455.4
617.5
-MS3(1267.1->778.7), 15.5min #1056
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 5
Peak 7
Peak 6
Peak 8
Fig. S4 (a)
1420.1
1352.9
-MS, 16.0min #1086
660.9840.4
1309.7
1026.3
-MS2(1352.9), 16.0min #1087
659.6
965.6
-MS3(1353.2->1026.4), 16.0min #1088
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
420.0 554.2 637.5 751.3 836.6 944.6
1351.8
1108.7
-MS, 16.2min #1098
458.5621.5
783.6 1116.6
945.7
-MS2(1108.7), 16.2min #1099
622.1
782.5
-MS3(1108.6->945.9), 16.2min #1100
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
1090.0
1309.0
-MS, 16.5min #1113
779.7
1025.7
1267.8
617.6
-MS2(1309.0), 16.5min #1114
455.5
-MS3(1308.7->617.5), 16.5min #1115
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
1258.0
1191.3
-MS, 16.9min #1142
881.71145.9
659.8
-MS2(1191.3), 16.9min #1143
204.8
455.5
631.5
1028.0
-MS3(1191.2->659.9), 16.9min #1144
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 9 Peak 10
Peak 11 Peak 12
Fig. S4 (a)
799.1996.2
1194.71578.2
919.4
-MS, 21.0min #1464
553.3 650.2 994.4 1361.7
781.7
-MS2(919.4), 21.1min #1465
620.5
715.5
-MS3(919.2->782.5), 21.1min #1466
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
661.5
935.6
1299.51559.8
825.5
-MS, 21.3min #1483
662.7
779.8
-MS2(825.5), 21.3min #1484
457.5
617.5
-MS3(825.4->781.3), 21.4min #1485
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
901.7
1565.3
-MS, 21.5min #1492
619.6
781.7
-MS2(1565.3), 21.5min #1493
457.5
619.5
-MS3(1565.1->780.9), 21.5min #1494
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
785.0901.9
1097.6
1565.5
-MS, 21.7min #1510
782.0
-MS2(1565.5), 21.7min #1511
457.5
619.5
-MS3(1565.2->781.5), 21.8min #1512
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 13 Peak 14
Peak 15 Peak 16
Fig. S4 (a)
901.7
1564.9
-MS, 22.0min #1525
619.7
781.9
-MS2(1564.9), 22.0min #1526
457.5
619.5
-MS3(1565.3->781.0), 22.0min #1527
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
899.9
1561.0
-MS, 22.5min #1559
617.7
779.8
-MS2(1561.0), 22.5min #1560
425.5
617.5
-MS3(1561.3->778.9), 22.5min #1561
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
827.51374.2
1560.9
1172.1
-MS, 24.0min #1673
617.8
780.1
-MS2(1172.1), 24.0min #1674
455.6
617.7
-MS3(1171.7->778.7), 24.1min #1675
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
618.7806.2
1237.5
-MS, 27.1min #1875
617.8
-MS2(1237.5), 27.1min #1876
437.4
571.5
-MS3(1237.2->618.7), 27.1min #1877
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 17 Peak 18
Peak 19 Peak 20
Fig. S4 (a)
Peak 21 Peak 22
660.0728.0793.8
1408.5600.1
-MS, 28.2min #1955
407.4
455.4
569.5
785.5 987.0
497.5
-MS2(600.1), 28.3min #1956
437.4
-MS3(600.3->497.8), 28.3min #1957
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
602.3 692.0761.2 840.2 986.7 1095.1
1408.4
-MS, 29.8min #2064
659.61282.11364.0
599.6
-MS2(1408.4), 29.8min #2065
391.3
455.5497.3
-MS3(1408.9->598.2), 29.9min #2066
0
50
100
Intens.[%]
0
50
100
[%]
0
50
100
[%]
200 400 600 800 1000 1200 1400 m/z
Fig. S4 (a)
317.4 537.9658.0730.9803.8 999.0 1107.0
1268.3
1430.2
1269.1
-MS, 14.2min #774
782.1944.2
1106.3
1271.1
-MS2(1269.1), 14.2min #7750
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
311.3385.3
576.0728.6 835.8
971.8
1106.1
1220.0
1345.0 1572.4
1107.1
-MS, 14.5min #792
457.7
619.8781.9
944.0
1119.8
-MS2(1107.1), 14.5min #7930
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
379.8458.8 606.2
687.8
801.1
956.1
1061.8
1430.1
-MS, 13.2min #716
0
20
40
60
80
100
Intens.[%]
200 400 600 800 1000 1200 1400 m/z
249.6 375.9 461.9 687.4926.7 1106.1
1268.3
1337.1
1269.1
-MS, 13.7min #746
620.0 782.2
944.3
1106.4
1271.1
-MS2(1269.1), 13.7min #7470
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 1 Peak 2
Peak 3 Peak 4
Fig. S4 (b)
249.3
419.9
487.8 597.8 806.1 902.5
1024.1
1106.1
1260.0
1107.1
-MS, 15.8min #865
619.8 781.9
944.0
1059.6
-MS2(1107.1), 15.9min #8660
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
444.0 553.5
1106.8
1220.4
1107.5
-MS, 16.0min #876
619.9 782.1
944.2
-MS2(1107.5), 16.1min #8770
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
317.8 506.0 686.6
836.3
1023.9
1268.2
1269.1
-MS, 15.3min #835
619.8 781.9
944.01106.0
1272.0
-MS2(1269.1), 15.3min #8360
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
243.3317.5 440.1 576.3 684.3756.2836.4912.2
1024.1
1106.2
1220.1
1354.1
1107.1
-MS, 15.6min #853
619.8 781.9
944.0
1113.6
-MS2(1107.1), 15.6min #8540
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 7 Peak 8
Peak 5 Peak 6
Fig. S4 (b)
317.5 495.5 589.8665.5786.3
944.4
1058.1
945.3
-MS, 16.4min #896
457.7
619.8781.9
897.5
-MS2(945.3), 16.4min #8970
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
249.6 399.3 521.9 651.3
797.1
1000.01067.9
865.6
-MS, 19.9min #1096
796.2
-MS2(865.6), 19.9min #10970
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
243.5 398.5 521.5 628.2
796.5
932.0 1320.9
864.2
-MS, 20.9min #1153
796.1
-MS2(864.2), 20.9min #11540
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
590.4
830.4
902.5
1070.1 1221.1 1320.8
1565.4
-MS, 21.3min #1176
619.8
782.1
1568.4
-MS2(1565.4), 21.3min #11770
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 11 Peak 12
Peak 9 Peak 10
Fig. S4 (b)
636.2 731.5
809.7
887.7
1290.5
1291.4
-MS, 22.0min #1214
633.8
1294.3
-MS2(1291.4), 22.0min #12150
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
317.7412.0 666.3 1299.6
823.9
-MS, 22.4min #1235
407.8
471.8615.9
762.0
-MS2(823.9), 22.4min #12360
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
666.1
-MS, 22.5min #1243
619.7
-MS2(666.1), 22.5min #12440
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
237.3 362.4441.6 553.5
618.1
710.8
779.8855.4
1083.2
1440.6
618.8
-MS, 23.1min #1276
241.0
409.6
467.6
625.6
675.9789.7
1067.7 1503.1
-MS2(618.8), 23.1min #12770
20
40
60
80
100
Intens.[%]
0
20
40
60
80
100
[%]
200 400 600 800 1000 1200 1400 m/z
Peak 15 Peak 16
Peak 13 Peak 14
Fig. S4 (b)
Fig. S5
Saponin aglycone molecular mass of 456(––); 458(––); 472(––)
14 16 18 20 22 24 26 2812 30
14* 15*
7
2
64*5
8*
13
9*6*
12 13*11 16
10*
14 16 18 20 22 24 26 2812 30
2*4*
5
67*
8 9
10
1112
13
14
15
1 3
14 16 18 20 22 24 26 2812 30
2A (EIC)
1 (TIC)
2 (TIC)
2B (EIC)
2C (EIC)
2D (EIC)
1
3*
46*
7*9
10*11*
8
12
1415*16
17*18*
19
20*21*
52
RT (min) RT (min) RT (min)
aLUP2G + CYP716A80 + UGT73C11
bLUP2P + CYP716A81 + UGT73C13
cLUP2P + CYP716A80 + UGT73C11
Figure S5. Saponins produced by expression of different combinations of B. vulgaris genes coding for OSCs, P450s and UGTs in N. benthamiana plant leaves. Panels (a-c) show LC-MS profiles for (1) (a) LUP2G, (b) and (c) LUP2P; (2) LUPs in combination with (a) CYP716A80, (b) CYP716A81, and (c) BvCYP716A80; (3) LUPs and CYPs in combination with (a) UGT73C11, (b) UGT73C13, and (c) UGT73C11. (2A) – (3D) EICs of (2) representing saponins with (2A) five sugar moieties (m/z 1266-1268, 1308-1310, 1380-1382, 1418-1420); (2B) four moieties (m/z 1088-1091, 1105-1110, 1145-1147, 1190-1192) (2C) two moieties (m/z 829-831, 901-903, 1564-1566) and (2D) one moiety (m/z 617-619, 666-668, 1236-1238, 1413-1415, 1408-1411). The y axis (ion count) of each chromatogram is scaled to the highest peak. Peaks highlighted with asterisk (*) correspond to saponins that significantly decreased or disappeared after sodium hydroxide-based saponification, indicating presence of sugar moieties at the C28 position of the triterpene aglycones. See Tables S5-S7 for more detailed information on detected saponins from these enzyme combinations.
