1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Supporting information
19
20
21
Figure S3. Protein sequence alignment of potato HQT and HCT with representative
members from other species. Accession numbers were as presented in Figure S2. Black
underlined bars represent the conserved motifs for the acyltransferase family members.
22
23
24
25
26
Figure S4. LCMS analysis of CGA isomers in tobacco leaves infiltrated with either
empty vector or StAN1. 3, 4, 5CGA, cis-45CGA and cis-5CGA, chlorogenic acid
isomers, respectively. The data represents the means ± SE (n=4). * indicates statistically
significant compared to WT based on t-test (p < 0.05). Refer to right Y-axis for all
underlined compounds.
27
28
29
30
31
Figure S5. The extent of HQT down-regulation in transgenic tubers. Relative
expression (arbitrary units) was calculated based on the expression of target genes as
compared to that of geometric mean of reference genes, elongation factor 1-α and actin.
The data represents the means ± SE (n=4). * indicates statistically significant compared
to WT based on t-test (p < 0.05).
32
33
34
35
36
37
Figure S6. Ion extraction chromatograms for representative individual phenolics from
tuber samples. WT and HQT transgenics are represented by solid and dotted lines,
respectively. (a) Peaks 1 to 5 represents chlorogenic acid isomers, 3CGA, cis-4CGA,
5CGA, 4CGA and cis-5CGA, respectively, (b) caffeic acid,(c) caffeoyl putrescine and (d)
Bis(caffeoyl)spermine1.
38
39
40
41
42
Figure S7. Levels of antioxidant activity in tubers of WT and the three transgenic lines
measured by the oxygen radical absorbance capacity (ORAC) and ferric reducing ability
of plasma (FRAP) assays. The data represents the means ± SE (n=4). * indicates
statistically significant compared to WT based on t-test (p < 0.05).
Figure S1. HPLC chromatogram of potato tuber methanolic extracts showing the A320
profile. The large peak is 5CGA, and some of the smaller peaks are CGA isomers. Inset
shows A360 of the same extract
Figure S2. Phylogenetic tree of HQT and HCT from different plant species. Phylogenetic
analyses were conducted in MEGA4. The evolutionary history was inferred using the
Minimum Evolution method. The percentage of replicate trees in which the associated
taxa clustered together in the bootstrap test (500 replicates) are shown. The evolutionary
distances were computed using the Poisson correction method and are in the units of the
number of amino acid substitutions per site. Accession numbers are ABA46756, StHQT
(Solanum tuberosum); ACF17657, CaHCT (Capsicum annuum); ACJ23164, CcHQT2
(Cynara cardunculusartichoke); AED95744, AtHCT (Arabidopsis thaliana); BAA87043,
IbHCT (Ipomea batatus); CAD47830, NtHCT (Nicotiana tabacum); CAE46932, NtHQT;
CAE46933, SlHQT (Solanum lycopersicum); CAM84302, CcHQT1;
PGSC0003DMC400024883, StHCT;. All sequences were from NCBI, except StHCT
which was from potato genome sequence consortium.
1
43
44
Figure S8. Levels of lignin in leaves and tubers of WT and the three transgenic lines. The
data represents the means ± SE (n=4).
45
46
47
48
49
50
51
52
53
54
55
Figure S9. Relative amounts of additional polyamines in WT and the three transgenic
lines. BCS1 (Bis(caffeoyl)spermine1) was from tubers, the
caffeoyl(dihydrocaffeoyl)spermidine isomers, CDS1 and CDS2, from flowers and BCS2,
BCS3 and CD (caffeoyl derivative) are from leaves. Relative amounts were calculated
based on the peak area of extracted ion chromatogram and normalized to sample weight
and dilution. The data represents the means ± SE (n=4). * indicates statistically
significant compared to WT based on t-test (p < 0.05).
56
57
58
59
60
61
62
63
Figure S11. Carotenoid metabolism in tubers of WT and the three transgenic lines.
Levels of (a) major and (b) minor carotenoids and (c) carotenoid gene expression. Total
carotenoid levels are shown in inset. CHY-b, β carotene hydroxylase; LCYe, lycopene ε
cyclase; PSY1 and 2, phytoene synthase isoforms; ZDS, ζ -carotene desaturase; ZEP,
zeaxanthin epoxidase. Relative expression (arbitrary units) was calculated based on the
expression of target genes as compared to that of geometric mean of reference genes,
elongation factor 1-α and actin. The data represents the means ± SE (n=4). * indicates
statistically significant compared to WT based on t-test (p < 0.05).
Figure S10. Levels of aromatic amino acids in WT and the three transgenic lines from
different tissues. (a)Tubers, (b) leaves and (c) flowers. The data represents the means ±
SE (n=4). * indicates statistically significant compared to WT based on t-test (p < 0.05).
64
65
2
Table S1. Protein similarity matrix of HQT and HCT from different species.
SlHQT NtHQT
StHQT
SlHQT
NtHQT
StHCT
NtHCT
94%
StHCT NtHCT
AtHCT
93%
73%
73%
73%
93%
75%
75%
74%
74%
75%
74%
96%
87%
87%
3
Table S2. Retention times and MS data of selected compounds present in potato phenolic
extracts separated by HPLC.
tR (m in) com pound
abbreviation
molecular
ion (m/z)
-
2.5
tyrosine
Tyr
180 [M-H]
4.0
phenylalanine
Phe
164 [M-H]
163, 119, 93
-
147, 148, 72
-
5.9
tryptophan
Trp
203 [M-H]
6.5
3-O -chlorogenic acid
3CGA
353 [M-H]
6.8
salicylic acid glucoside
SAG
299 [M-H]
7.0
caffeoyl putrescine
CP
251 [M+H]
7.6
cis -4-chlorogenic acid
cis -4CGA
353 [M-H]
159, 116, 142, 186
-
191, 179, 135
-
137
+
-
5-O -chlorogenic acid
5CGA
353 [M-H]
8.5
4-O -chlorogenic acid
4CGA
353 [M-H]
8.6
bis(dihydrocaffeoyl)spermine
BDCS
531 [M+H]
8.7
caffeic acid
CA
179 (M-H)
9.1
cis -5-chlorogenic acid
cis -5CGA
353 [M-H]
9.2
bis(caffeoyl)spermine 1
BCS1
527 [M+H]
191,179, 161,135
-
173, 179, 191, 135
+
-
-
9.6
bis(dihydrocaffeoyl)spermidine
BDCSD
474 [M+H]
9.8
bis(caffeoyl)spermine 2
BCS2
527 [M+H]
10.3
caffeoyl(dihydrocaffeoyl)spermidine 1 CDS1
472 [M+H]
10.4
feruloyl quinic acid1
FQA1
367 [M-H]
10.6
caffeoyl(dihydrocaffeoyl)spermidine 2 CDS2
472 [M+H]
+
+
+
+
11.5
12.0
feruloyl quinic acid2
tris(dihydrocaffeoyl)spermine
FQA2
TDCS
367 [M-H]
+
695 [M+H]
12.1
bis(caffeoyl)spermine 3
BCS3
527 [M+H]
12.3
quercetin 3-O -rutinoside-glucoside
QRG
771 [M-H]
12.6
quercetin-diglucoside
QDG
625 [M-H]
caffeoyl derivative
13.4
kaempferol-3-O -rutinoside-glucoside KRG
755 [M-H]
13.5
tris(dihydrocaffeoyl)spermidine
TDCSD
638 [M+H]
13.6
quercetin xylose-rutinoside
QXR
741 [M-H]
14.0
quercetin 3-O -rutinoside
RUT
609 [M-H]
14.5
kaempferol 3-O -rutinoside
CD
KMP
530 [M+H]
+
+
+
-
593 [M-H]
293, 222, 513, 165, 367
107, 75
191, 179, 173
+
-
13.2
234, 163, 89, 115
173, 179, 135, 191
-
8.3
MS/MS (m/z )
291, 509, 220, 163
222, 457, 165, 310, 236
291, 509, 220, 163
222, 310, 165, 455
161, 135, 335
222, 310, 165, 455
179, 135, 191, 161, 143
293, 222, 531, 474, 457
291, 509, 220, 163
591, 300, 301, 609
301, 445
368,510.5, 292.5
575, 285, 593
456, 474, 293, 222
301, 609, 591, 475
301, 271, 343
285, 195, 239
4
Table S3. Levels of additional individual anthocyanins in different tissues WT and the three
transgenic lines from different tissues.
Tubers
DeCRG
PeCRG
PtCR
WT
T3
T10
T16
MFRG
13.93
12.14
13.03
11.03
± 1.15 11.8 ± 0.98 10.56 ± 0.67
± 2.03 13.5 ± 2.50 11.80 ± 1.54
± 2.39 8.4 ±5361455673425
1.29 9.68 ± 1.34
± 2.57 9.2 ± 1.68
1674894113123
10.15 ± 1.46
Leaves
Lines
WT
T3
T10
T16
PtR
PtD
µg g-1
Lines
DRG
22.19
23.63
42.2*
31.33
27.6
47.7*
43.2*
47.3*
065924646422
± 3.14 5.08 79206494539
± 0.61 DL
993215053533
± 2.44 2.64* ±917507567901
0.29 0.63* ±5101591407599
0.37
986276840434
± 1.89 2.86* ±046952839875
0.33 0.95* ±6092568236514
0.08
368338500156
± 3.54 2.60* ±028618168635
0.52 1.02* ±940451295066
0.09
Flowers
PlDGG
± 1.97 5.60 ± 1.89
± 2.47 4.87 ± 0.92
±287374214681
6.78 10.6 ± 2.70
± 4.43 8.78 ± 1.40
DeCRG
186.5
187.2
373*
282*
PeCRG
PtCR
± 19.1 31.8 530260793313
± 2.16 11.5 ± 217859052054
1.55
± 15.2 63.5* 263475474608
± 2.18 23.2* ±61.45
22947329132
±.38217113178
18.4 72.0* ±569123656406
4.34 24.5* ±948024551444
2.29
±.288418554449
16.2 73.2* ±41.97
06138651827
25.1* ±5 1.95
84999261014
DeCRG, PeCRG, 3-(coumaroyl) rutinoside-5-glucosides of delphinidin and pelargonidin,
respectively; DRG, delphinidin rutinoside-5-glucoside; MFRG, malvidin 3-(feruloyl) rutinoside5-glucoside; PlDGG, pelargonidin 3-diglucoside-5-glucoside; PtCR, petunidin coumaroyl
rutinoside; PtR, petunidin rutinoside; PtD, petunidin derivative. DL, below detectable levels. The
data represents the means ± SE (n=4). * indicates statistically significant compared to WT based
on t-test (p < 0.05).
Table S4. Retention times and MS data of anthocyanins in potato extracts separated by HPLC.
molecular ion
tR (m in) com pound
abbreviation
3.5
petunidin 3-rutinoside-5-glucoside
PtRG
[M] (m/z)
787
4.6
delphinidin 3-rutinoside-glucoside
DRG
773
303,611,465
5.2
malvidin 3-rutinoside-5-glucoside
MRG
801
639,331,493
6.1
pelargonidin 3-diglucoside-5-glucoside
PlDGG
757
271,595
6.3
petunidin-3-rutinoside
PtR
625
317,479,302
8.8
delphinidin-3-(coumaroyl) rutinoside-5-glucoside DeCRG
919
303,757,465
9.06
petunidin 3-(caffeoyl) rutinoside-5-glucoside
PtCfRG
949
787,317,479
10.3
petunidin 3-(coumaroyl) rutinoside-5-glucoside
PtCRG
933
317,771,479
11.1
petunidin 3-(feruloyl) rutinoside-5-glucoside
PtFRG
963
317,801,479
11.4
peonidin 3-(coumaroyl) rutinoside-5 glucoside
PeCRG
917
755,301,463
11.8
malvidin 3-(coumaroyl) rutinoside-5-glucoside
MCRG
947
785,331,493
12.3
petunidin-3-(coumaroyl) rutinoside
PtCR
771
317,479,245
12.5
malvidin 3-(feruloyl) rutinoside-5-glucoside
MFRG
977
331,815,493
13.1
petunidin derivative
PtD
801
317, 480, 549
+
MS/MS (m/z )
625,317,479
5
Table S5. Retention times and UV-spectral data of the compounds present in potato carotenoid
extracts separated by HPLC.
t R (m in) com pound
abbreviation
λ m ax (nm )
8.9
neoxanthin
Neo
415 439 467
9.9
violaxanthin
Viola
415 438 467
11.6
cis -neoxanthin
Crt1
412 435 463
12.4
neochrome
Crt2
398 420 447
16.4
antheraxanthin
Anthera
422 443 472
17.7
lutein
Lutein
421 444 472
18.9
zeaxanthin
Zea
424 449 478
20.7
cis -α-carotene isoform Crt3
417 438 469
21.4
cis -α-carotene isoform Crt4
410 437 466
22.2
5,6-epoxy-α-carotene
Crt5
416 439 469
26.2
β-carotene
b car
424 450 477
27.0
cis -β-carotene
Crt6
424 442 470
6
Table S6. List of primers used in this study.
Primer
Primer sequence
Potato genome Contig
Accession
PAL-F
PAL-R
C4H-F
C4H-R
HCT-F
HCT-R
C3H-F
C3H-R
HQT-F
HQT-R
CHS-F
CHS-R
DFR-F
DFR-R
AN1-F
AN1-R
bHLH1-F2
bHLH1-R2
PSY1-F
PSY1-R
PSY2-F
PSY2-R
CHY2-F
CHY2-R
ZEP-F
ZEP-R
LCY-e-F
LCY-e-R
ZDS-F
ZDS-R
StCO1-F
StCO3F
StCO1/3-R
SP3D-F
SP3D-R
SP3A,B,5G-F
SP3A-R
SP3B-R
SP5G-R
ACGGGTTGCCATCTAATCTGACA
CGAGCAATAAGAAGCCATCGCAAT
CCCAGTTTTTGGAAATTGGCTTCA
GCCCCATTCTAAGCAAGAGAACATC
CCCGAATGCAGATACTGTTCCTGA
AGTGAGTCCTCGTGCCATACAAGT
TTGGTGGCTACGACATTCCTAAGG
GGTCTGAACTCCAATGGGTTATTCC
CCCAATGGCTGGAAGATTAGCTA
CATGAATCACTTTCAGCCTCAACAA
CACCGTGGAGGAGTATCGTAAGGC
TGATCAACACAGTTGGAAGGCG
TCACAGGAGCAGCTGGATTTATCG
TCAGGATCACGAACAGTAGCATGG
AAGTATGGGCAAGCCAATGCCA
GCCCACCATTGCATATCGTTGTTGTC
TCTCTTGGAYGGTGTAGTGGAACT
AACAGCGGATGACGGAGTATTGCT
CGGTCTGCTATTGTKGCTACTCC
CAGGAACAGGTATGTCTGGCTTC
AGGTGGGAGACAGGRGTGGAATTT
TGATGACACAGCCATTTCTCCAGC
GCCTATTGCCAACGTGCCTTACTT
TCCAACTCTTCAAGCCCTCCTACT
TTGCACTATCGGGTCTGTCTCACA
CCAGGTGCCATGTTCGCTTTGTAA
GCAAAATGGATGTGGCAG
CAATGTTGCACCAGTAGGATCAG
TTGCCATGTCAAAGGCCA
ACAGGCACTCCGACCAATTT
GACAGTTGCCATTCCGCAACAT
AAGTCTGGTGGCTTCTCGTCACAA
AGGCACAAAGTGAGGCAGCAT
ATCCTCTYGTTGTTGGTCGTGT
CAACTTCAACCCTTGGCTGGTT
ACACTCTGRTTRTGGTGGATCCTG
CTGACGCCAATTTGGTGCATAA
TTCGGGAGCATGGACAATTTCTCG
GCCCAATTGCTGGAACAACACGAA
PGSC0003DMS000001020
BG887005
PGSC0003DMS000000597
BG889948
PGSC0003DMS000000559
AJ507825
PGSC0003DMS000002734
BQ514440
PGSC0003DMS000000585
BF154152
PGSC0003DMS000001347
BG888147
PGSC0003DMS000000741
AY289921
PGSC0003DMS000000121
PGSC0003DMT400033569
PGSC0003DMS000001131
TC122598
PGSC0003DMS000002349
L23424
PGSC0003DMS000000374
HM013963
PGSC0003DMS000002087
ADF28630
PGSC0003DMS000001425
AF321537
PGSC0003DMS000001954
TC114158
PGSC0003DMB000000004:961826..966450
PGSC0003DMB000000004:972177..975380
PGSC0003DMB000000142
PGSC0003DMT400041725
PGSC0003DMT400041726
PGSC0003DMB000000512
7
mAU
A320
mAU
800
3000
A360
700
600
500
2500
400
300
200
2000
100
0
0
2
4
6
8
10
12
14
16
18
min
1500
1000
500
0
0
2
4
6
8
10
12
14
16
18
min
Figure S1
StHQT
100
100
SlHQT
100
NtHQT
IbHCT
CcHQT1
100
CcHQT2
AtHCT
NtHCT
100
0.05
StHCT
100
89
CaHCT
Figure S2
8
Figure S3
20
*
empty
StAN1
0.2
*
15
10
*
*
0.1
mg g-1 dw
mg g-1 dw
0.3
5
*
0.0
0
cis-4CGA cis-5CGA
3CGA
4CGA
5CGA
Figure S4
9
HQT Relative expression
0.06
0.04
* * *
0.02
0
WT T3 T10 T16
Figure S5
Figure S6
10
µmole TE g-1 dw
240
40
* * *
* * *
30
160
20
80
10
0
0
ORAC
FRAP
Figure S7
11
mg g-1 dw
24
16
8
0
Tuber
Leaf
Figure S8
Relative abundance
6
WT
4
***
2
T3
**
*
T10
T16
*
**
*
*
*
*
**
*
*
0
BCS1
CDS1
CDS2
BCS2
BCS3
CD
Figure S9
12
a
1.2
mg g-1 dw
WT
T3
0.8
T10
T16
*
0.4
*
0
b
0.6
mg g-1 dw
Tyrosine
0.3
Phenylalanine
Tryptophan
*
***
0
Tyrosine
c
Phenylalanine
Tryptophan
0.6
mg g-1 dw
**
0.3
*
*
0
Tyrosine
Phenylalanine
Tryptophan
Figure S10
13
a
2.0
5
µg g-1 dw
4
3
2
1.0
1
*
0
*
**
0.0
Viola
b
Lutein
Anthera
Neo
b car
Zea
0.2
µg g-1 dw
WT
T3
T10
T16
0.1
*
**
0.0
Crt1
Crt2
Crt3
Crt4
Crt5
Crt6
PSY1
PSY2
ZDS
LCY-e
ZEP
CHY-b
c
Relative expression
0.04
0.02
0
Figure S11
14