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BMC Biotechnology

BioMed Central

Open Access

Research article

Glycosyltransferase efficiently controls phenylpropanoid pathway
Anna Aksamit-Stachurska2, Alina Korobczak-Sosna1, Anna Kulma1 and
Jan Szopa*1
Address: 1Faculty of Biotechnology, Wroclaw University, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland and 2Faculty of Biological Sciences,
Wroclaw University, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland
Email: Anna Aksamit-Stachurska - anna.aksamit@interia.pl; Alina Korobczak-Sosna - alina1k@interia.pl;
Anna Kulma - kulma@ibmb.uni.wroc.pl; Jan Szopa* - szopa@ibmb.uni.wroc.pl
* Corresponding author

Published: 5 March 2008
BMC Biotechnology 2008, 8:25

doi:10.1186/1472-6750-8-25

Received: 27 September 2007
Accepted: 5 March 2008

This article is available from: http://www.biomedcentral.com/1472-6750/8/25
© 2008 Aksamit-Stachurska et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: In a previous study, anthocyanin levels in potato plants were increased by
manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content
and tuber yield were dramatically reduced in the transgenic plants, which over-expressed
dihydroflavonol reductase (DFR).
Results: Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently
transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in
order to obtain plants with a high anthocyanin content without reducing tuber yield and quality.
Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural
substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the
super-transformed plants, tuber production was much higher than in the original transgenic plants
bearing only the transgene coding for DFR, and was almost the same as in the control plants. The
anthocyanin level was lower than in the initial plants, but still higher than in the control plants.
Unexpectedly, the super-transformed plants also produced large amounts of kaempferol,
chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins.
Conclusion: In plants over-expressing both the transgene for DFR and the transgene for UGT,
the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a
novel approach to manipulating phenolic acids synthesis in plants.

Background
The phenylpropanoid pathway is the source of numerous
phenylalanine derivatives. The main branches of the phenylpropanoid pathway are presented in Figure 1. The
main products of the various branches are lignin, flavonoids, chlorogenic acid, salicylic acid and catecholamines.
Of the compounds synthesized by the phenylpropanoid
pathway, the most abundant is lignin, which makes up
66.0% of the total, followed by proanthocyanins

(18.6%), phenolic acids (13.0%), anthocyanins (2.5%),
salicylic acids (0.4%), and catecholamines (0.4%). Most
of the compounds synthesized by the phenylpropanoid
pathway can be glycosylated by glycosyltransferases. Glycosylation increases solubility, reduces reactivity, and
increases stability.
The components of this pathway play an important role in
plant physiology. Flavonoids represent a large class of
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Figure of
Scheme 1 the phenylpropanoid pathway
Scheme of the phenylpropanoid pathway. L-phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonol
synthase (FS), glucosyltransferase (GT), hydroxylase-O-methyl transferase (HOT), methyltransferase (MT), anthocyanin synthase (AS), and leucoanthocyanidin reductase (LCR).

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phenylpropanoids. Flavonoids serve as pigments, which
attract pollinators and protect the plant from UV irradiation. Flavonoids also act as antioxidants, photoreceptors,
antimicrobials, feeding deterrents, and metal chelators
[1].
The phenylpropanoid pathway begins with the deamination of phenylalanine to cinnamic acid. In subsequent
reactions, hydroxycinnamoyl CoA thioester is formed.
This compound is the substrate for branch pathways
responsible for the synthesis of lignin monomers,
anthocyanins, coumarins and chlorogenic acid [2].
Among the products of the phenylpropanoid pathway are
flavanones, which are converted to kaempferol or
anthocyanidins in several steps involving hydroxylation.
These products are then glycosylated by a specific glycosyltransferase. In potatoes, the two main anthocyanins are
pelargonidin and peonidin, which have a tri-saccharide
side chain attached to the 3-hydroxy group of the aglycone [3].
In a previous study, anthocyanin levels in potato plants
were modified by manipulating genes connected with the
flavonoid biosynthesis pathway [3]. The genes manipulated were those coding for the key enzymes in the pathway: chalcone synthase (CHS), chalcone isomerase
(CHI), and dihydroflavonol reductase (DFR). The plants
were transformed using constructs containing one, two or
all three of these genes. The constructs contained the
cDNA sequences inserted in either the sense or anti-sense
orientation. The selected transgenic plants were then analyzed for anthocyanin content and antioxidant capacity.
The construct that was most effective in increasing
anthocyanin synthesis was the single-gene construct containing the cDNA sequence for DFR in the sense orientation. The construct which was most effective in repressing
anthocyanin biosynthesis was the single-gene construct
containing the cDNA sequence for DFR in the anti-sense
orientation. In the plants carrying the transgene for DFR,
anthocyanin content was perfectly correlated with antioxidant capacity. However, in all of the transgenic plants,
starch content was lower than in the control. Depending
on the construct used, starch content was reduced from a
few percent to over 90%. The reduction in starch content
was accompanied by a significant reduction in tuber yield.
This suggests that high concentrations of flavonoids suppress carbohydrate synthesis, thereby reducing tuber production.
Glycosylation reduces the reactivity and increases the stability of flavonoids. Recently, transgenic potato plants
which over-produce glycosyltransferase (UGT) have been
produced and characterized [4]. In tuber extracts from
plants which over-express the gene for UGT, the level of

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3,5-O-substituted anthocyanidins was significantly higher
than in tuber extracts from the control plants, but significantly lower than in tuber extracts from plants bearing the
transgene for DFR. Of the six anthocyanins identified, the
ones present in the highest amounts were pelargonidin 3rutinoside-5-glucoside acylated with p-coumaric acid and
peonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid. Glycosylated flavonoids do not reduce starch
content and tuber yield.
In order to substantially increase flavonoid content without reducing tuber yield and quality, plants over-expressing the transgene for DFR were super-transformed with
cDNA coding for the UGT of Solanum sogarandinum under
the control of the tuber-specific B33 promoter.
It was expected that the level of anthocyanin synthesis
would be at least the same as in the initial plants, and that
starch content and tuber yield would not be reduced
because of the higher levels of anthocyanins. In the supertransformed plants, tuber production was much higher
than in the initial plants bearing only the transgene coding for DFR, and was almost the same as in the control
plants. The anthocyanin level was lower than in the initial
plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced
large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins. In plants
over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted
away from the anthocyanin branch. This represents a
novel approach to manipulating phenolic acids synthesis
in plants. Chlorogenic acid is a strong antioxidant which
reduces the oxidation of low-density lipids and may prevent carcinogenesis. Increasing dietary intake of chlorogenic acid should therefore be beneficial to consumer
health.

Results
The substrate specificity
The glycosyltransferase gene used in this study was isolated from several cold-induced clones of the cold-resistant potato species Solanum sogarandinum [5]. The product
of this gene was 83% similar to the flavonoid 7-O-glycosyltransferase of Nicotiana tabacum, 68% similar to the
anthocyanin-5-O-glycosyltransferase of Petunia hybrida,
60% similar to the UGT of Perilla frutescens, 57% similar
to the UGT of Verbena hybrida, and 53% similar to the
UGT of Torenia hybrida. A molecular phylogenetic tree
based on the deduced amino acid sequence of the UGTs
of several plant species is presented in Figure 2.

In order to study the expression of UGT, the cDNA encoding for it was inserted into E. coli using the pQE 30 vector.
The recombinant protein was purified by affinity chroma-

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Mlecular2phylogenetic tree based on deduced amino acid sequences of plant glycosyltransferases
Figure
Mlecular phylogenetic tree based on deduced amino acid sequences of plant glycosyltransferases. Amino acid
sequences of plant glycosyltransferases were obtained by using Blast program with Blosum62 algorithm. The tree was constructed by the Fast Minimum Evolution method. The dendrogram was created using the Clustal Sequence Alignment software
package.

tography and subjected to enzymatic analysis. As stated
above, the UGT of S. sogarandinum is highly similar to the
flavonoid 7-O-glycosyltransferase of N. tabacum and the
anthocyanin-5-O-glycosyltransferase of P. hybrida. Therefore, the recombinant protein was tested using several
compounds that serve as substrates for both the tobacco
and petunia enzymes.
Of the substrates tested, (cinnamic acid, caffeic acid, coumaric acid, kaempferol, kaempferol-3-O-glycoside,
kaempferol-5-O-glycoside, kaempferol-7-O-glycoside, 5deoxykaempferol, peonidin chloride and peonidin-3-Oglycoside) kaempferol was the substrate most effectively
glycosylated by the recombinant protein (Fig. 3a, 3b). The
products of the reaction were separated and identified
using HPLC or UPLC. In a previous study [4], this enzyme
was predicted to be anthocyanin 5-O-glycosyltransferase.
However, further enzyme studies revealed it to be a flavonoid 7-O-glycosyltransferase that also had anthocyanin 3and 5-O-glycosyltransferase activity. The activity of the
recombinant protein on various phenolic compounds is

presented in Figure 3c. With cinnamic acid and coumaric
acid, the reaction rate was one-fourth the rate with kaempferol. With caffeic acid and chlorogenic acid, only trace
activity was observed. Kinetic parameters were calculated
based on the Lineweaver-Burk plot using kaempferol as
the substrate (Table 1). The Km was 8.5 μM for kaempferol, and 21.4 μM for peonidin. This indicates that the
recombinant protein is a 7-O-glycosyltransferase. In
another study, the 5-O-glycosyltransferase from Dorotheanthus bellidiformis was highly homologous to the glycosyltransferases of several solanaceous plants and could
also glycosylate flavonols such as quercetin [6]. This suggests that the natural substrates of the UGT of S. sogarandinum include both anthocyanidins and flavonols such as
kaempferol and quercetin.
Generation and selection of transgenic plants
In order to produce transgenic potato plants with
increased levels of stable glycosylated anthocyanins, twoweek-old leaf explants of transgenic potato line DFR 11
[3] were subsequently super-transformed by dipping

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a

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b
P-3-O -glu

1

1

2

2
P

3

3

P-3-O -glu

P

c

Figure of
Analysis 3 specificity of recombinant UGT
Analysis of specificity of recombinant UGT. a) Analysis of specificity of recombinant UGT by UPLC, 1.: Standards of
kaempferol glucosides and kampferol. 2.:Standard of kaempferol 3.: Chromatogram of standard kaempferol incubated with
recombinant glycosyltransferase. Incubation with recombinant UGT, UDP-glucose, and keampferol produced a major product
with the expected retention time and UV spectrum of keampferol-7-O-glucoside. The enzyme assay and product analysis is
described in the Methods section. b) Analysis of specificity of recombinant UGT by HPLC. 1.: Standard of peonidin-3-O-glucoside, 2.: Standard of peonidin chloride., 3.: Chromatogram of standard peonidin chloride incubated with recombinant glycosyltransferase. Incubation with recombinant UGT, UDP-glucose, and peonidin chloride produced a major product with the
expected retention time and UV spectrum of peonidin-3-O-glucoside. The enzyme assay and product analysis is described in
the Methods section. c). Substrate specificity of recombinant UGT. The substrate specificity was measured as described in the
Methods section with the use of UDP-Glu as a glucose donor. The mean value (n = 4) ± SE is presented.

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Table 1: Substrate specificity of recombinant UGT.

Substrates

Km (μM)

Vmax (μmol/s × mg)

Vmax/Km

peonidin
Kaempferol

21.42 ± 1.52
8.5 ± 0.62

478.00 ± 138.12
268.685 ± 36.31

22.35
31.36

The substrate specificity was measured as described in the Methods
section with the use of UDP-Glu as a glucose donor. The mean value
(n = 4) ± SE is presented.

them in a suspension of Agrobacterium tumefaciens carrying the cDNA sequence coding for the UGT of S. sogarandinum (GenBank accession number AY033489). This
transgene was placed under the control of the tuber-specific B33 promoter. The regenerated plants were then
screened using PCR with primers specific for the gene for
dihydroflavonol 4-reductase and for glycosyltransferase
(Fig. 4a). Those with the highest signal were selected for
further analysis. Further selection was carried out using
northern and western blotting. Expression of both transgenes was high in all of the lines selected (Fig. 4b,c). The
selected super-transformed lines were subjected to further
selection based on the activity of both DFR and UGT (Fig
4d). DFR activity in the super-transformed plants was as
high as it was in line DFR 11. This indicates that the second round of transformation had no effect on DFR activity. UGT activity in the super-transformed plants was
measured using kaempferol as the substrate. In lines DFR/
UGT 42, 45, 46, 47 and 48, UGT activity was from 20% to
50% higher than in the control. These lines were therefore
selected for further analysis.
Phenotypical analysis
In the super-transformed plants, the above-ground parts
of the plant were generally the same in size and shape as
in line DFR 11 and the control. The only exception was
flower color, which was white in the super-transformed
plants. This suggests either that they contained less
anthocyanins, or that they accumulated colorless compounds such as leucoanthocyanins. In line DFR 11, tuber
production is lower than in the control [3]. In all the
super-transformed plants, tuber production was increased
compared to DFR 11 and was about the same as in the
control except in line 48, in which it still remained significantly lower (Fig. 5a). The tuber size was also about the
same as in the control in lines 42 and 47 while in lines 45
and 46 it was larger. In contrast, like DFR 11, the line 48
produced smaller tubers than the control (Fig. 5b). The
number of tubers per plant was the same as the control in
lines 42 and 47, and lower than the control in lines 45, 46,
and 48. Nevertheless, this was still higher than in the single DFR transformant (Fig. 5c). This suggests that the overexpression of the gene coding for UGT cancels out the
reduction in yield caused by the over-expression of the
gene coding for DFR.

Anthocyanins content in tuber extracts
The levels of flavonoids in the tubers of the super-transformed plants was also measured using thin layer chromatography and were found to be higher than in the control
plants (Fig. 6a). Anthocyanins content was also measured
using HPLC in order to determine the effect of the two
transgenes. As expected, elevated levels of anthocyanins
were detected in the super-transformed plants (Fig. 6b).
The anthocyanins found in the highest amounts were the
trisaccharide derivatives of pelargonidin (pelargonidin 3rut 5-glu acylated with p-coumaric acid), peonidin (peonidin 3-rut 5-glu acylated with p-coumaric acid) and malvidin (malvidin 3-rut 5-glu acylated with p-coumaric
acid). This confirmed the results obtained by TLC analysis. The level of pelargonidin glycoside was significantly
higher in two super-transformed lines than in the control.
In line 46, the level was more than twice as high as in the
control. In line 42, the level was 1.5 times higher than in
the control. Nevertheless, this was a reduction compared
to the pelargonidin level found in line DFR 11, in which
it was more than three times higher than in the control. In
the other super-transformed lines, the level of pelargonidin glycosides was also higher than in the control,
although the difference was not statistically significant.
The malvidin content was slightly but insignificantly
higher in lines 42, 45 and 46 than in the control. The level
of peonidin glycoside was higher than the control in only
two of the super-transformed lines. In lines 42 and 47, the
level of peonidin glycoside was about 1.3 times higher
than in the control but the changes were not statistically
significant. Considering that UGT is highly homologous
to flavonoid 7-O-glycosyltransferase, the levels of
anthocyanin precursors were also measured. Kaempferol
content was more than four times higher in lines 42, 45,
46, and 47 and almost four times higher in line 48 than in
the control. Except of line 48, this was an almost two-fold
increase in the amount of kaempferol in the super-transformed lines compared to DFR 11 (Fig. 6b). This suggests
that 7-O-glycosylated kaempferol is a less suitable substrate for DFR than non-glycosylated kaempferol. The
increase in UGT activity therefore increased flavonol accumulation, thereby suppressing anthocyanin synthesis.
This might explain why the amount of accumulated
anthocyanins in the super-transformed lines was less than
half as much as in line DFR 11.
Phenolic acid, lignin and proanthocyanins content in tuber
extract
In plants, chlorogenic acid is synthesized using three different pathways. In the glycosylation pathway, the first
step is the glycosylation of the carboxyl group of cinnamic
acid [7]. The product of this reaction is then converted
into an activated intermediate, caffeoyl D-glucose. This
intermediate is then combined with quinic acid to yield
chlorogenic acid. Therefore, the levels of phenolic acids in

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C

DFR

42

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45

46

47

48
C

DFR

42

45

46

47

48

cDNA
DFR

a

C

DFR

42

45

46

47

cDNA
UGT

C

48

b

DFR

42

45

46

47

48
mRNA
UGT
28 S
rRNA

mRNA
DFR
28 S
rRNA

C

DFR 42

45

46

47

48

C

DFR

42

45

46

47

48

DRF
Rubisco

c

UGT
Rubisco

Activity of UGT
%

%

Activity of DFR
200

150

150

100

100
50

50
T
45
*
D
FR
/U
G
T
46
*
D
FR
/U
G
T
47
*
D
FR
/U
G
T
48
*

G

42
*
D
FR
/U

D
FR
/U
G
T

D
FR
*
FR
/U
G
T
42
D
*
FR
/U
G
T
45
D
*
FR
/U
G
T
46
D
*
FR
/U
G
T
47
D
*
FR
/U
G
T
48
*
D

C

D
FR

0

0

C

d

Figure 4
Selection of transgenic plants
Selection of transgenic plants. a) Agarose gel electrophoresis of PCR products obtained with primers for DFR (1143 bp)
cDNA and UGT (1100 bp) cDNA on genomic DNA isolated from tissue-cultured potato plants. C: negative control (nontransformed plant). DFR: transgenic plant over-expressing DFR. Transgenic lines over-expressing DFR and UGT are numbered. b) Northern blot analysis of RNA isolated from tubers of control plant (marked C), tubers of transgenic plant overexpressing DFR and tubers of independent transgenic lines over-expressing DFR and UGT (numbered). 50 μg of total RNA
was loaded onto each lane, and the first blot was hybridized with 32P- labeled DFR cDNA and the second blot was hybridized
with 32P- labeled UGT cDNA Bottom panel shows ribosomal RNA stained with ethidium bromide as a control of RNA applied
onto the gel. c) Western blot analysis of protein extract isolated from the potato tuber. Blots were probed with anti DFR antibodies (left) and anti UGT antibodies (right). C: negative control (non-transformed plant). DFR: transgenic plant over-expressing DFR. Transgenic lines over-expressing DFR and UGT are numbered. Bottom panel is the ribulose-1,5 bisphosphate
carboxylase/oxygenase (Rubisco) on the same blot stained with Pounceau red. d) Activity of UGT and DFR in transgenic and
wild type potato plants DFR and UGT over-expressing plants (numbered-striped bars) were analyzed and compared with
transgenic plant over-expressing DFR (DFR- black bar) and with the control (C- grey bar). Asterisks (*) indicate values that are
significantly different from the wild type plants with p > 0.05.

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the super-transformed lines were measured and found to
be significantly higher in most lines than in the control
(Fig. 7a). The level of isochlorogenic acid was as much as
six times higher than in the control, as was the case with
line 46. The level of chlorogenic acid was two to three
times higher than in the control. The level of caffeic acid
was 1.3 times higher than in the control. When assayed in
vitro, the recombinant protein had a low affinity for cinnamic acid. Nevertheless, the affinity in vivo may be high
enough to ensure adequate synthesis of chlorogenic acid
by the glycosylation pathway.

a
1000

FW of tubers per plant (g)

900
800
700
600
500
400
300
200
100

FR
*
FR
/G
T
42
D
FR
/G
T
45
D
FR
/G
T
46
D
FR
/G
T
D
47
FR
/G
T
48
*

D

C

0

D

b
70

Fw of single tuber (g)

60
50
40
30
20
10

48
*

47
D
FR

/U

G
T

G
T

46
*

/U

G
T
/U

D
FR

D
FR

G
T
/U

/U

D
FR

D
FR

25

45
*

42

D
FR
D
FR

c

G
T

C
C

*

-

Number of tuber per plant

Phenolic acids serve as substrates in lignin biosynthesis.
Lignin is composed of monomer precursors called monolignols. The main step in lignin formation is the conversion of cinnamic acid into p-coumaryl alcohol, coniferyl
alcohol and sinapyl alcohol. In the super-transformed
lines, the level of sinapic acid glycoside was higher than in
the control in all analyzed lines except line 48. Nevertheless, lignin content was not higher (Fig. 7b).

20
15
10
5

48
*

47
D
FR

/U

G
T

G
T

/U

D
FR

D
FR

/U

G
T

46
*

45
*
G
T

/U
D
FR

D
FR

/U

G
T

42

*

-

The super-transformed plants had white flowers, which
suggest that colorless compounds were accumulated. The
first product of DFR activity is leucoanthocyanidin, which
is colorless. Leucoanthocyanidin is subsequently converted to proanthocyanin, which is also colorless. This
conversion is effected by an enzyme, which is similar to
DFR acting together with glycosyltransferase [8]. Therefore, the level of proanthocyanins in the super-transformed plants was measured and found to be higher in all
of the super-transformed lines than in the control (Fig.
7c). The differences were statistically significant except in
lines 46 and 48. In line 45, the level of proanthocyanins
was 31% higher than in the control. In line 46, the level
of proanthocyanins was only 7% higher than in the control. On the other hand, in line DFR 11, the level of proanthocyanins was 22% lower than in the control. This
indicates that the expression of the gene for UGT changes
the flux of substrates and precursors in the phenylpropanoid pathway.

Figure 5
The yield of field cultivated transgenic and control plants
The yield of field cultivated transgenic and control
plants. Tubers from 10 plants were collected and used for
analysis. Tubers from independent transgenic lines overexpressing DFR and UGT (numbered) were analyzed and
compared to the control (C) and to transgenic line overexpressing DFR (DFR) from field trials performed in 2005.
Grey bar- control, black bar- transgenic plant over-expressing DFR, striped bars- transgenic lines over-expressing DFR
and UGT. Asterisks (*) indicate values that are significantly different from the wild type plants at p > 0.05.

Antioxidant capacity in tuber extracts
Antioxidant compounds enable plants to cope with
adverse environmental conditions, including low temperatures, UV radiation, microbial infection, and infestation
by pests. Among the antioxidant compounds found in
plants are phenolic acids such as chlorogenic acid, isochlorogenic acid, caffeic acid, and anthocyanins. All of
these compounds help protect plants from environmental
stress. They also help maintain oxidative status in potato
tubers [3]. Therefore, the total antioxidant capacity was
measured in tuber extracts from the super-transformed
plants. Results were recorded in terms of IC50, which represents the amount of extract required to inhibit luminol
luminescence by 50%. The total antioxidant capacity was

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a)

caffeic acid
chlorogenic
acid
anthocyanidins

DFR/UGT 46

DFR/UGT 47

Malvidin

48
T

D

FR
/U
G
D

FR
/U
G

T

T

47

46

45
FR
/U
G
D

Kaempferol

48
*
T

47
*
G
/U
FR
D

D

FR

/U

/U

G

G

T

T

46
*

45
*
T
FR
D

D

FR

/U

/U

G

G

T

D

FR

42
*

*

15
10
5
0
C

mg/100g DW
D
FR
*
D
FR
/U
G
T
42
D
FR
/U
G
T
45
D
FR
/U
G
T
46
D
FR
/U
G
T
47
D
FR
/U
G
T
48

C

mg/100g DW

Peonidin
100
80
60
40
20
0

T

42
T
FR
/U
G

D

FR

C

D

D

mg/100g DW

10
8
6
4
2
0

D
FR
D
*
FR
/U
G
T
42
*
D
FR
/U
G
T
45
D
FR
/U
G
T
46
*
D
FR
/U
G
T
47
D
FR
/U
G
T
48

150
100
50
0

DFR/UGT 48

FR
/U
G

DFR/UGT 45

FR

DFR/UGT 42

Pelargonidin

C

mg/100g DW

b)

DFR

D

C

Figure 6
Determination of anthocyanins content in epidermal tuber extracts
Determination of anthocyanins content in epidermal tuber extracts. a) TLC chromatogram of separated fractions of
anthocyanidins from epidermal tuber extracts of control (C), transgenic plant over-expressing DFR (DFR) and transgenic lines
over-expressing DFR and UGT (numbered). As a mobile phase 1-butanol-acetic acid-water (4:1:5) was used. b) HPLC determination of anthocyanins content in epidermal tuber extracts from control (C-grey bar), transgenic plant over-expressing DFR
(DFR-black bar) and transgenic lines over-expressing DFR and UGT (numbered-striped bars). The mean value (n = 6) ± SE is
presented. Asterisks (*) indicate values that are significantly different from the wild type plants at p > 0.05.

from 60 to 98% higher in the super-transformed lines
than in the control (Fig. 8a). The correlation coefficient
for IC50 and phenolic acids content was -0.82. The correlation coefficient for IC50 and anthocyanins content was

-0.72. This suggests that phenolic acids and anthocyanins
affect antioxidant capacity to about the same degree.

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Chlorogenic acid

Caffeic acid

48
*

47
*

G
T

D

D

FR

FR

/U

/U

G
T

G
T

46
*

45
*

D

D

FR

FR

/U

/U

G
T

FR
D

DF

FR

R/
U

/U

G
T

D

C

48
*

47
*

G
T

G
T
R/
U
DF

DF

DF

R/
U

R/
U

G
T

G
T

46
*

45
*

42
*

R

G
T

DF

R/
U
DF

42
*

450
400
350
300
250
200
150
100
50
0

mg/100g DW

400
350
300
250
200
150
100
50
0
C

mg/100g DW

a)

Isochlorogenic acid
mg/100g DW

400
300
200
100

Sinapic acid

2500

50

2000

mg/100g DW

1500
1000
500
0

40
30
20
10

c)

48

*

*

G
T
/U

/U
DF
R

DF
R

G
T

47

46

*
DF
R/
U

G
T

45

*
/U
G
T

42
DF
R

DF
R

/U
G
T

/U
FR
D

D

DF
R

48
G
T

G
T

47

46
/U

G
T
FR

/U

G
T
FR
D

D

D

FR

FR

/U

/U

45

42
G
T

FR
D

C

0
C

mg/100g DW

48

/U
FR

D

Lignins

b)

G
T

47
*

46
*
/U
FR
D

D

FR

/U

G
T

G
T

45
*
G
T

FR
D

D

FR

/U

/U

G
T

D

C

FR

42
*

0

48
G
T

/U
FR

D

D

FR

/U

G
T

47
*

46
G
T

/U

/U

FR
D

G
T
D

FR

/U

G
T

42
*

*
FR
D
FR
D

45
*

700
600
500
400
300
200
100
0
C

mg/100mg DW

Proanthocyanins

Figure
Phenolic7acid, lignin and proanthocyanins content in tuber extract
Phenolic acid, lignin and proanthocyanins content in tuber extract. a) HPLC determination of phenolic acid in epidermal tuber extracts from control (C-grey bar), transgenic plant over-expressing DFR (DFR-black bar) and transgenic lines overexpressing DFR and UGT (numbered-striped bars). The mean value (n = 6) ± SE is presented. Asterisks (*) indicate values that
are significantly different at p > 0.05 from the wild type plants. b) Determination of lignin and sinapic acid content in epidermal
tuber extracts from control (C-grey bar), transgenic plant over-expressing DFR (DFR-black bar) and transgenic lines overexpressing DFR and UGT (numbered-striped bars). The mean value (n = 6) ± SE is presented. Asterisks (*) indicate values that
are significantly different (with p > 0.05) from the wild type plants. c) Determination of proanthocyanins content in epidermal
tuber extracts from control (C-grey bar), transgenic plant over-expressing DFR (DFR-black bar) and transgenic lines overexpressing DFR and UGT (numbered-striped bars). The mean value (n = 6) ± SE is presented. Asterisks (*) indicate values that
are significantly different from the wild type plants at p > 0.05.

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Antioxidative protection of polyunsaturated fatty acids by
tuber extracts
Flavonoids and phenolic acids are powerful antioxidants.
This suggests that they may prevent lipid oxidation both
in vivo and in vitro. They may therefore improve the storability and quality of oil [9].

a
IC 50
DFR/UGT 48*
DFR/UGT 46*
DFR/UGT 42*
C
0

20

40

60

80

ug/ml

0,5
0,45
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0

TBARS

D
FR

C
D
FR
/U
*
G
D
FR T 4
2*
/U
G
D
FR T 4
5*
/U
G
D
T
FR
46
/U
*
G
D
FR T 4
7*
/U
G
T
48
*

Discussion

L

mol/kg

b

Therefore, tuber extracts from the super-transformed
plants were tested in terms of their ability to prevent peroxidation of polyunsaturated fatty acids in flaxseed oil.
Lipid peroxidation was evaluated by measuring the level
of thiobarbituric acid-reactive substances (TBARS). In
extracts from the super-transformed lines, the level of
TBARS was from 60% to 85% percent lower (Fig. 8b). This
is about twice as much as with the extract from line DFR
11. The correlation coefficient for antioxidant capacity
and phenolic acids content was -0.70. The correlation
coefficient for antioxidant capacity and anthocyanins content was -0.71.

Antioxidant potential of potato tuber extract
Figure 8
Antioxidant potential of potato tuber extract. a) The
antioxidant potential (presented as IC50) of potato extract
from the control (C), DFR- transgenic plant over-expressing
DFR and transgenic lines over-expressing DFR and UGT
(numbered). The analysis of potato extracts was performed
as specified in Methods. Grey bar- control, black bar- transgenic plant over-expressing DFR, striped bars- transgenic
lines over-expressing DFR and UGT. The mean values from
three independent measurements + confidence interval is
presented. Asterisks (*) indicate values that are significantly
different at p > 0.05 from the wild type plants. b) TBARS formation in Linola flax oil in the presence of potato extract.
Crude oil extracted from Linola seeds alone or supplemented with potato extract from the control (C), transgenic
plant over-expressing DFR (DFR) and transgenic lines overexpressing DFR and UGT (numbered) was heated for 40 min
at 140°C. Data are mean TBARS levels from six repetitions
(two independent lipid extractions) measured spectrophotometrically at 535 nm. White bar- oil extracted from Linola
seeds alone, grey bar- oil supplemented with extract of control, black bar- oil supplemented with extract of transgenic
plant over-expressing DFR, striped bars- oil supplemented
with extract of transgenic lines over-expressing DFR and
UGT. Asterisks (*) indicate values that are significantly different (with p > 0.05) from the wild type plants.

Secondary metabolites are very important constituents of
plant cells. The phenylpropanoid pathway is the source of
large number of phenylalanine derivatives [1,10]. Deamination of phenylalanine by phenylalanine-ammonia
lyase yields trans-cinnamic acid, which is then hydroxylated to give 4-coumarate. This is the starting point for several very important biochemical pathways in plants. In
potatoes, the main products of the various branches of the
phenylpropanoid pathway are lignin, flavonoids, chlorogenic acid, salicylic acid and catecholamines. Lignin, flavonoids and phenolic acids are the most abundant
products. Lignin synthesis increases in response to microbial infection. Lignin is the second most abundant macromolecule in the biosphere, surpassed only by cellulose.
Lignin enhances pressure resistance in plant cell walls
[11]. Interest in flavonoids and phenolic acids is increasing because some of them have been found to have antioxidant, anti-allergenic, anti-viral or anti-inflammatory
properties [12,13].
Flavonoids protect plants from UV irradiation [14]. They
also chelate metals. Phenolic acids are the most powerful
antioxidants found in plants [15]. Increasing the activity
of the phenylpropanoid pathway in crop plants may
prove useful in increasing resistance to biotic and abiotic
stressors. It may also help in producing fruits and vegetables with higher levels of antioxidants, which would
increase storability and nutritional value.
Genetic transformation is a powerful method for improving crop plants. In recent studies, we have generated transgenic plants that over-express genes coding for key
enzymes involved in flavonoid synthesis. In one of these
studies, anthocyanin production was found to be greatly

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increased in transgenic potato plants that over-express
DFR [3]. However, the increase in anthocyanin content
was accompanied by a significant decrease in starch content and tuber yield. High levels of anthocyanins may
therefore inhibit carbohydrate synthesis. The reactivity of
anthocyanins can be reduced by glycosylation. In one
study on potatoes, over-expression of UGT increased the
level of glycosylated anthocyanidins [4]. In the present
study, transgenic potato plants bearing the cDNA
sequence for DFR were super-transformed with the cDNA
sequence for UGT. It was expected that this would substantially increase flavonoids content without severely
reducing starch content and tuber yield. The UGT of S. sogarandinum is highly homologous to enzymes found in
other plants, in particular the flavonoid 7-O-glycosyltransferase of N. tabacum and the anthocyanin-5-O-glycosyltransferase of P. hybrida. The chief substrate of
flavonoid 7-O-glycosyltransferase is kaempferol [16].
However, the specificity of a recombinant protein cannot
be reliably predicted on the basis of sequence homology
alone.
In this study, the activity of the recombinant UGT was
therefore tested in vitro using different substrates. The
recombinant UGT proved to glycosylate a spectrum of
substrates. However, it was more effective in glycosylating
flavonols such as kaempferol than in glycosylating
anthocyanidins. Based on enzyme kinetics, the recombinant UGT preferentially glycosylates the 7-OH group of
flavonols. Unexpectedly, the level of glycosylated
anthocyanidins was lower in the super-transformed lines
than in line DFR 11. This may be due to competition for
substrate, or it may be due to changes in the flow of compounds in the different branches of the phenylpropanoid
pathway, a phenomenon which has been recently
reported [17]. In the tubers of elicitor-treated potatoes,
decreased synthesis of chlorogenic acid is accompanied by
increased synthesis of products from other branches of the
phenylpropanoid pathway, for example, hydroxycinnamoyltyramine and feruloyltyramine. In another study,
on the other hand, manipulating hydroxycinnamoyl-CoA
quinate hydroxycinnamoyl transferase affected the level
of chlorogenic acid without affecting the levels of other
soluble phenolics [7].
The level of kaempferol and its derivatives was also measured in tuber extracts of the super-transformed lines. The
level of kaempferol glycoside was four times higher than
in the control. The glycosylation of kaempferol therefore
increased its accumulation and decreased its flux to further steps of anthocyanin biosynthesis.
Of the three pathways involved in the synthesis of phenolic acids, the glycosylation pathway is the most effective. Although the recombinant enzyme had a very low

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affinity for cinnamic acid in vitro, the levels of phenolic
acid were very high in tuber extracts from the super-transformed lines. The level of chlorogenic acid was up to three
times higher than in the control. This suggests that the
affinity of the recombinant UGT for cinnamic acid in vivo
is high enough to ensure adequate chlorogenic acid synthesis. The genes coding for the key enzyme of the glycosylation pathway are of particular interest because of the
high bioavailability of chlorogenic acid.
Based on studies using transgenic plants, the key enzyme
of the glycosylation pathway was identified as hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase
(HQT). In transgenic tomato plants, which over-express
the gene for HQT, the level of chlorogenic acid is up to
85% higher than in control plants [7]. In the present
study, the level of chlorogenic acid in the super-transformed plants was even higher, three times higher than in
the control. The regulation of anthocyanin biosynthesis
appears to be similar to the regulation of proanthocyanin
biosynthesis. This conclusion is based on several lines of
evidence:
• in extracts of sainfoin (Onobrychis viciifolia), dihydroflavonol reductase (DFR) and leucoanthocyanidin reductase
(LR) are active as a multi-enzyme complex [18];
• in barley, DFR is inhibited by leucoanthocyanidin [19];
• in transgenic Lotus corniculatus plants carrying the Sn
gene from maize, decreased proanthocyanidin levels were
accompanied by decreased DFR and LR activities [20]; and
• grains carrying a mutation in the Ant 19 gene accumulate approximately 10% wild type proanthocyanin and
have dramatically reduced DFR and LR activities [19].
Leucoanthocyanins, the immediate product of DFR, are
therefore converted primarily to proanthocyanins when
the glycosylation pathway is stimulated by the overexpression of UGT.
Compared to the control, antioxidant capacity was
induced to about the same degree both in the super-transformed lines and in line DFR 11. However, lipid peroxidation was more effectively prevented by tuber extracts from
the super-transformed lines than by the extract from line
DFR 11.
As expected, glycosylated intermediates of the phenylpropanoid pathway were less biologically active than their
non-glycosylated counterparts, and therefore did not
reduce starch content (data not shown) and tuber yield. In
the super-transformed lines, starch content and tuber
yield were about the same as in the control.

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Simultaneous over-expression of the genes for DFR and
UGT increases biosynthesis of kaempferol, anthocyanins
and phenolic acids. However, the increase in flavonols is
mostly due to the specificity of UGT, which prefers
kaempferol as a substrate. Further study is needed to elucidate why the increase in phenolic acids content is much
greater in transgenic plants bearing the genes for both
DFR and UGT than in transgenic plants bearing the gene
for either of these enzymes alone.

strain C58C1: pGV2260 [22]. Integration of the plasmid
was verified by restriction enzyme analysis. Explants of
young leaves of wild-type potato (S. tuberosum L. cv. Desiree) were transformed by immersing them in a suspension
of A. tumefaciens. The inoculated leaf explants were then
transferred to callus induction medium and shoot regeneration medium containing kanamycin (100 μg/ml). All
selection media contained cefotaxime in order to eliminate any remaining Agrobacterium cells [22].

Conclusion

Transgenic plant selection
The transformants were preselected by PCR and then
selected by means of northern and western blot analysis.

In transgenic plants simulanious expression od dihydroflavonol reductase and glycosyltransferase resulted in significant increase in flavonols and anthocyanins content.
The 4-fold increase in kaempferol content was primarily
due to the glycosyltransferase specificity

Methods
Plant material and bacterial strains
Potato plants (Solanum tuberosum L. cv. Desiree) were
obtained from "Saatzucht Fritz Lange KG" (Bad Schwartau, Germany). Control and transgenic plants were grown
in the greenhouse in soil under 16 h light (22°C) and 8 h
darkness (16°C) regime. Plants were grown in individual
pots and were watered daily. Tubers were harvested after
four months of growth and subsequently used for a field
trial conducted near Wroclaw between May and September, 2005. All data presented in this paper pertain to
tubers from field grown plants.

Escherichia coli strain DH5α (Bethesda Research Laboratories, Gaithersburg, USA) was cultivated using standard
techniques [21]. Agrobacterium tumefaciens strain C58C1
containing plasmid pGV2260 [22] was cultivated in YEB
medium [23].
Generation of transgenic plants
In this study, two types of transgenic plants were used:

DFR 11, which over-expresses the P. hybrida cDNA encoding dihydroflavonol 4-reductase (EMBL/GenBank database acc no. X15537); and
DFR/UGT, which is a DFR 11 transgenic line [3] supertransformed with recently cloned glycosyltransferase
cDNA from S. sogarandinum (EMBL/GenBank database
acc no. AY033489).
Leaf explants were transformed using the pBin vector containing the appropriate cDNA in sense orientation. The
cDNA was under the control of 35S promoter in DFR
plants, or the tuber specific B33 promoter in DFR/UGT
plants. In both lines, the cDNA was under the control of
the OCS terminator. The nptII gene was used as a selection
marker. The vector was introduced into the A. tumefaciens

Two-step PCR pre-selection was carried out with the following specific primers:
dihydroflavonol 4-reductase (DFR):
forward: GGTGCATTCTCTTTGCCACTTGC
reverse: GACAGTTTGCGTCACTGGAGCTG
glycosyltransferase (UGT):
forward: GTCCTCTTGGTGACATTTCCCACAC
reverse: TGAGGAAATGCCACCACAGGTACAC.
Genomic DNA isolated from three-week-old tissue-cultured plants was used as a template. Transgenic lines that
tested positive for the predicted PCR products were subject to further analysis. The predicted products were 1143
bp long for the DFR gene, and 1100 bp long for the UGT
gene. Final selection was carried using northern and western blot analysis. Total RNA was prepared from frozen
young plant leaves or tubers using the guanidinium
hydrochloride method [24]. The RNA was separated by
electrophoresis on 1.5% (w/v) agarose containing 15%
(v/v) formaldehyde. Then the RNA was transferred to a
nylon membrane (Hybond N, Amersham, UK). The
membrane was hybridized overnight at 42°C with radioactively labeled DFR cDNA and UGT cDNA as a probe.
Then the filters were washed three times with SSPE buffer
containing 0.1% SDS for 30 min at 42oC.
The fourteen transgenic lines that showed the highest levels of mRNA for UGT and DFR were finally selected by
immunodetection. Potato tubers were homogenized in
liquid N2. Total protein was extracted with buffer E containing 100 mM HEPES/NaOH (pH 7.4), 10 mM MgCl2,
2 mM EDTA (pH 8.0), 2 mM EGTA (pH 8.0), 1 mM PMSF,
0.2% Triton X-100, glycerol and 14 mM beta-mercaptoethanol. After centrifugation, proteins concentration
was measured using the Bradford method (Bio-Rad Pro-

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tein Microassay). The protein extracts prepared from the
potato tubers were separated on a 12% SDS-polyacrylamide gel and electrophoretically transferred onto a nitrocellulose membrane (Schleicher and Schuell). Following
transfer, the membrane was incubated with blocking
buffer (5% dry milk), and then with antibody against 5UGT-recombinant protein (1:2000 dilution) or with antibody against DFR-recombinant protein (1:3000 dilution). Goat anti-rabbit IgG conjugated with alkaline
phosphatase was used as a secondary antibody at a dilution of 1:1500. The alkaline phosphatase was then stained
with NBT (p-nitro blue tetrazolium chloride) and BCIP
(5-bromo-4-chloro-3-indoyl-phosphate p-toluidide).
DFR enzyme assay
Protein extracts were prepared from the potato tubers.
Potato tubers were homogenized in liquid N2. Total protein was extracted with buffer E (100 mM HEPES/NaOH
(pH 7.4), 10 mM MgCl2, 2 mM EDTA (pH 8.0), 2 mM
EGTA (pH 8.0), 1 mM PMSF, 0,2% Triton X-100, glycerol,
14 mM beta-mercaptoethanol).

The extracts were centrifuged at 15,000 g for 15 min at
4°C, and the supernatant was used for further analysis.
Protein concentration was measured using the Bradford
method. DFR activity was measured in accordance with
the procedure described by De-Yu [25]. The reaction mixture contained 25 mM Tris·HCl (pH 7.0), 4 mM NADPH,
100 μM taxifolin (dihydroquercetin), and protein extract.
The reaction was initiated by addition of NADPH at 25°C,
followed by measuring the rate of NADPH oxidation at
340 nm. The enzyme activity was calculated by using the
absorption coefficient of NADPH (6.22 mM-1·cm-1). One
unit of enzyme activity was equivalent to the oxidation of
1 μmol of NADPH per min.

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sis. Compounds were identified and quantified using
standards.
Transgenic plants were selected using an assay mixture
containing kaempferol as the substrate and tuber extract
(30 μg) as the enzyme source.
Extraction of phenolic compounds
150 mg of vacuum-dried tuber epidermis was extracted
with 1 ml methanol-1% HCl solution in an ultrasonic
bath for 15 min. After centrifugation supernatant was filtered through millipore (0.2 μm) then dried in a speedvac. Polyphenols were re-suspended in 1 ml methanol.
The solution was then applied onto SPE column (Merck,
Darmstadt, Germany). Retained compounds were eluted
from the solid phase with 40% MeOH and analyzed on
HPLC/UV.
HPLC analysis of phenolics
Phenolics were analyzed using an HPLC system (Knauer,
Germany) equipped with an automated sample injector
and a UV detector (Knauer variable wavelength monitor
type 87.00). The system was connected to a personal computer (HPLC Software/Hardware Package Version 2.21A).
The sample extract was separated on a LiChroCART® 2504 100 RP-18 (5 μm) column preceded by a LiChroCART®
4-4 100 RP-18 (5 μm) pre-column (Merck, Darmstadt,
Germany). Compounds were detected by on-column
measurement of UV absorption at 325 nm. The sensitivity
was set at 0.04 a.u.f.s. The flow rate was adjusted to 1.0
ml/min. Phenolics were separated using a mobile phase
consisting of the following components:

A: acetonitrile-formic acid (90:10 v/v); and
B: water-formic acid (90:10 v/v).

UGT enzyme assay
UGT activity was determined using the following standards as acceptor substrates: cinnamic acid, caffeic acid,
coumaric acid, kaempferol, kaempferol-3-O-glycoside,
kaempferol-5-O-glycoside, kaempferol-7-O-glycoside, 5deoxykaempferol, peonidin chloride and peonidin-3-Oglycoside (TransMIT, Marburg, Germany). UDP-Glu was
used as the donor of the sugar moiety. The reaction mixture (total volume, 75 μl) used for the assay of glycosyltransferase activity consisted of increasing content of
aglycones (from 2 to 15 μg per sample, 1 mg/ml stock
solution in ethylene glycol monomethyl ether) as the substrate, PBS buffer (pH 8.0), and 9 μl of UDP-Glu (10
nmol/μl). The reaction was carried out for 16 min at 30°C
and was started by adding 50 μl (1 mg/ml) of enzyme
solution. The phenolic acid and anthocyanin were then
extracted with 500 μl of methanol, dried, re-dissolved in
methanol (50 μl) and subjected to HPLC or UPLC analy-

For the first two minutes, isocratic elution was carried out
using 10% A in B. From 2 to 25 minutes, a linear gradient
was applied using 10 to 30% A in B. From 25 to 27 minutes, a linear gradient was applied using 30 to 70% A in B.
The volume of the sample injected was 20 μl. Calibration
graphs of polyphenols were prepared by measuring the
areas under the peaks. The graphs were linear in the range
examined (0.02 to 0.10 mg/ml). For the HPLC analyses,
gradient grade acetonitrile was used. Water was glass distilled and deionized. Solvent solutions were vacuum
degassed with sonication before use. All experiments were
performed at room temperature (20°C). Compounds
were identified and quantified using standards.
Analysis of the substrate specificity of recombinant UGT
by UPLC
The products of the reaction of recombinant UGT with
different substrates were analyzed using the Acquity UPLC

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system (Waters). A 2 μl sample was applied to an Acquity
UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). The
mobile phase was passed through the column at a flow
rate of 0.4 ml/min. The mobile phase consisted of the following components:
A: 50 mM aqueous ammonium formate (pH 3.0); and
B: 100% acetonitrile.
The column was eluted for 1 min with 80% A in B, for 4
min with 60% A in B, and for 6 min with 80% A in B. The
column was kept at room temperature. A photodiode
array (PDA) was used to detect UV visible absorption
between 210 and 500 nm. Compounds were identified
and quantified using standards.
Determination of antioxidant capacity
Antioxidant activity in the extracts was measured using
chemiluminescence. 10 mg of potato tuber epidermis was
extracted with methanol. The extract was diluted from
1,000 to 15,000 times with water and directly analyzed.
The experiments were performed in a final volume of 250
μl on white microplates in a freshly prepared solution
containing 0.1 M Tris-HCl buffer (pH 9.0) and 4 mM
AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride). The luminol solution (100 μM) and diluted extracts
were automatically injected. The photons produced in the
reaction were counted on an EG&G Berthold LB96P
microplate luminometer at 30°C. The antioxidant potential was defined as the amount of tuber extract that inhibits luminol chemiluminescence by 50% and was
expressed as IC50.
Determination of proanthocyanins
The method used was based on the fact that condensed
tannins give rise to anthocyanins when heated in mineral
acid. Dried tuber peels (100 mg) were extracted with 7 ml
of n-BuOH-conc. HCl (95:5, v/v) and 0.2 ml of a 2% (w/
v) NH4Fe(SO4)2 × 12 H20 in 2 M HCl. The mixture was
incubated at 95°C for 40 min. After centrifugation, the
absorbance at 550 nm measured. Proanthocyanin content
was calculated using catechin as standard (A1% at 550 nm
= 280).
TBARS determination
The level of thiobarbituric acid-reactive substances
(TBARS) in the samples was determined [26]. Oil samples
(1 μl) were oxidized at 140°C for up to 40 min with or
without (2 μl) potato extract (10 mg vacuum-dried tuber
epidermis was subjected to acidic-methanol extraction).
Two ml of reagent A (15% trichloroacetic acid and 0.37%
thiobarbituric acid in 0.25 M HCl) was added and the
mixture was thoroughly blended. Test tubes containing
the samples were stopped with glass marbles, heated at

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100°C for 15 min, cooled under running tap water, and
centrifuged for 10 min at 2,000 g. The absorbance was
measured at 535 nm using a spectrophotometer (Cecil
CE-2020). The reference blank contained the TBA reagent.
The oil from flaxseeds used for reaction was prepared in
accordance with the method described by Allen and Good
[27]. Briefly, 1 g of seeds was ground in a mortar with 1
ml of water. The homogenate was suspended in 2 ml of
methanol and 4 ml of chloroform. 3.5 ml of 0.9% NaCl
was then added. The mixture was gently agitated and left
to settle for 24 h at room temperature. The lower chloroform phase was collected and the extraction was repeated.
The chloroform was evaporated using a rotary vacuum
evaporator. The lipids were resuspended in chloroform/
methanol (1:2 v/v) and stored at -20°C.
Analysis of lignin content
10 mg of vacuum-dried tuber epidermis was suspended in
10 ml of water and heated for 60 min at 65°C. The sample
was then filtered through a GF/A glass fiber filter and
sequentially rinsed in water, ethanol, acetone and diethyl
ether. The filter was then placed in a glass vial and heated
overnight at 70°C. 2.5 ml of 25% acetyl bromide was
added, and the vial was heated for 2 h at 50°C. After cooling of the samples 10 ml of 2 N sodium hydroxide and 1
ml of acetic acid was added. After incubation overnight at
room temperature, the absorbance at 280 nm was measured. A calibration curve was constructed using coniferyl
alcohol as the standard. Results were expressed in terms of
equivalents of coniferyl alcohol.
Statistical analysis
Data on the parameters measured were statistically elaborated using ANOVA and the Laven test, followed by the
RIR Tukey or Kruskal-Wallis test. All calculations were carried out using the STATISTICA 7.1 software package
(StatSoft Polska, Poland).

Authors' contributions
AAS constructed and selected the transgenic plants, and
performed the experiments and the statistical analysis.
AKS participated in the HPLC analysis. AK carried out the
UPLC analysis and helped draft the manuscript. JS conceived of the study, and participated in its design and
coordination. All authors read and approved the final
manuscript.

Acknowledgements
This study was supported by grants 2P06A 02029 and PBZ-MNiI-2/1/2005
from the Polish Ministry of Science and Higher Education.

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