Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera)

Article abstract of DOI:10.1021/jf035234r

Stilbenes are phenolic molecules that have antifungal effects in the plant and antioxidant and anti-cancer effects when consumed in the human diet. Glycosylation of stilbenes increases their solubility and may make them more easily absorbed by the intesti

Full text of DOI:10.1021/jf035234r

J. Agric. Food Chem. 2004, 52, 3467−3472 3467
Polyphenol Glucosylating Activity in Cell Suspensions of Grape
Vitis vinifera)
(
MARK N. KRASNOW* AND TERENCE M. MURPHY
Section of Plant Biology, Division of Biological Sciences, University of California,
One Shields Ave., Davis, CA, 95616
Stilbenes are phenolic molecules that have antifungal effects in the plant and antioxidant and anti-
cancer effects when consumed in the human diet. Glycosylation of stilbenes increases their solubility
and may make them more easily absorbed by the intestine. We have found an activity in extracts of
cultured cells of Vitis vinifera (cv. Gamay Freaux) that glucosylates the stilbene resveratrol to form
piceid. The Km for UDP-Glucose was 1.2 mM, and the Km for resveratrol was 0.06 mM, values simi-
lar to those of other phenolic glucosyltransferases. We investigated the resveratrol glucosylating activity
of the enzyme extracted from cells grown under different light treatments (dark, visible light, light +
ultraviolet (UVC) radiation) and found the activity to be unaffected or slightly reduced. In contrast,
UVC light strongly stimulated extractable quercetin glucosyltransferase activity. These results, com-
bined with analysis of phenolic compounds extracted from the differently treated cells, suggest that
the resveratrol glucosyltransferase is distinct from the glucosyltransferase(s) active on other phenolics.
KEYWORDS: Vitis vinifera; stilbene; resveratrol; glucosyltransferase
INTRODUCTION
stilbenoids. Glycosylation also increases the solubility of
lipophilic molecules and allows for their storage within the plant.
Cell suspension cultures of Vitis Vinifera (cv. Gamay Freaux)
isolated from berry pulp fragments produce several stilbenoids.
The primary stilbenoid accumulated by these cells is piceid
Stilbenoids are polyphenolic secondary metabolites produced
in several plant families, including Vitaceae, Arachaceae, and
Pinaceae. They are synthesized by the enzyme stilbene synthase
(STS), which combines one molecule of hydroxycinnamoyl-
(
resveratrol-O-â-glucoside), though resveratrol is also ac-
CoA and three molecules of malonyl-CoA, yielding resveratrol
as a product.
Stilbenoids are thought to play a phytoalexin role in the plants
that produce them. Their accumulation can be induced by path-
ogen attack (1), UV light (2), and methyl jasmonate treatment
cumulated to a lesser extent. Other studies using these cells have
also identified the presence of pterostilbene, picetannol, the
viniferins, and astringin (15-18). Only astringin is a glucoside;
the others differ from resveratrol either by added hydroxyl
groups (picetannol), methylation of hydroxyls (pterostilbene),
or condensation (viniferins).
(3, 4). Stilbenes inhibit the germination and growth of fungal
mycelia of Botrytis cinerea, Plasmopara Viticola, and Cla-
dosporium cucumerinum in Vitro (5-8). Wheat and barley (non
stilbenoid producing plants) transformed with a stilbene synthase
gene showed increased resistance to fungal pathogens (9).
The enzyme UDP-glucose/flavonoid glucosyltransferase
(UFGT) is capable of glycosylating anthocyanidins, producing
the stable anthocyanins. We here report the extraction and
characterization of an enzyme activity, analogous to that of
UFGT, that glycosylates resveratrol to produce piceid (Figure
Stilbenoids have antioxidant properties in vitro (10) and have
been shown to inhibit events in cancer initiation, promotion,
and progression (11). Grapes and grape products constitute the
greatest source of stilbenoids in the human diet (12). Stilbenoids,
along with other phenolic compounds, may be responsible for
the inverse relationship between moderate wine consumption and
the occurrence of coronary heart disease and cancer (13). To act
as antioxidants, these chemicals must be absorbed into the blood-
stream; glycosylation may make phenolics more easily absorbed
by the intestine. Glycosylated quercetin (a flavonol) was absorb-
ed more than twice as well as the aglycone in human ileostomists
1
).
MATERIALS AND METHODS
Plant Material. Cell cultures were established from the flesh of a
Vitis Vinifera (cv. Gamay Freaux) berry in 1978 by Dr. J. C. Pech
(
ENSA, Toulouse, France) and have been subcultured continuously
since then. The growth medium consists of 20 g/L sucrose, 250 mg/L
casein hydrolysate, macronutrients (19), micronutrients (20), vitamins
(21), 0.2 mg/L Kinetin (0.93 µM), and 0.1 mg/L R-naphthalene acetic
acid (0.54 µM). Cells were grown in 25 mL of media in 125 mL
Erlenmeyer flasks. Cells were subcultured weekly at a 1:10 (v:v) ratio
into new media and were incubated at 25 °C under continuous rotary
shaking at 100 rpm (the standard growth conditions). Visible light
treatment was constant irradiation from two F20 cool white fluorescent
(14); this may also be true of other phenolics, including
*
To whom correspondence should be addressed. E-mail: mnkrasnow@
ucdavis.edu. Mailing Address: Plant Biology Section, University of
2
California, One Shields Avenue, Davis, CA, 95616. Fax: (530) 752-5410.
bulbs, at 30-cm distance (30 µmol photons/m ). Dark treatments
1
0.1021/jf035234r CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/04/2004

3468 J. Agric. Food Chem., Vol. 52, No. 11, 2004
Krasnow and Murphy
Figure 2. â-Glucosidase digestion of cell extract and resveratrol
glucosylation product. A is the resveratrol glucosyltransferase reaction
product. B is the reaction product treated with glucosidase. C is phenolic
cell extract. D is cell extract treated with glucosidase Samples were
incubated overnight at room temperature before HPLC analysis. The
retention time (X-axis) for piceid is 51 min, and for resveratrol is 63 min.
The peak at 55 min in the cell extract is due to an anthocyanin acylated
with a hydroxycinnamic acid. Y-axis is absorbance at 316 nm.
Figure 1. Reactions catalyzed by glucosyltransferases in grape cells. A
is the UFGT reaction using cyanidin, producing cyanidin-3-glucoside. B
is the UFGT reaction using quercetin, producing quercetin-3-glucoside.
C is the resveratrol glucosylation reaction, producing piceid.
temperature. Products were extracted with ethyl acetate and analyzed
as described above.
Photoisomerization of Piceid and Resveratrol. A 750-µL aliquot
of cell extract and 500 µL of 125 mM resveratrol were irradiated
overnight at 12 °C using a tungsten halogen lamp (1300 µmol photons/
m -s). Duplicate samples were kept in darkness. Phenolics were
analyzed by HPLC as previously described.
involved covering culture flasks with foil to exclude light. The ultra-
violet (UV) treatment was a 10 min pulse of UVC (unfiltered mercury
vapor lamp producing primarily 254 nm radiation) in a sterile hood
applied to continuously shaken light-grown cultures on day six of
growth. All experimental samples were harvested on day seven of
culture.
2
RESULTS AND DISCUSSION
Enzyme Extraction. All steps were carried out at 4 °C. Enzymes
were extracted from vacuum filtered cells (3 g) by freezing them in
liquid nitrogen and grinding them in the presence of glass beads (1.5
g) and PVPP (3 g). A 10-mL aliquot of the extraction buffer (0.2 M
sodium phosphate (pH 6.8), 5 mM EDTA, and 1mM DTT) was added
to the ground cells, and the resulting slurry was centrifuged at 12 000g
for 20 min to remove insoluble material. The supernatant was collected
and subjected to ammonium sulfate precipitation. The 30-80%
ammonium sulfate pellet was resuspended in 500 µL of extraction buffer
and dialyzed overnight against the same buffer. Samples were then
assayed immediately or frozen at -20° C. Protein concentrations were
estimated by the method of Bradford (22) with bovine serum albumin
as a standard.
Resveratrol Glucosyltransferase Characterization. The cell
enzyme extract was capable of glycosylating resveratrol,
producing piceid. The identity of the reaction product was
verified by its co-chromatography with the major stilbenoid
extracted from the cells, which has been shown by other studies
to be piceid (3, 16). In confirmation, when cell phenolic extracts
and resveratrol glucosylation products were subjected to â-glu-
cosidase digestion, resveratrol was produced (Figure 2). The
resveratrol glucosylation reaction was linear with time up to 5
h of incubation at 35 °C and was linear with enzyme concentra-
tion up to 250 and 500 µg protein (data not shown). Reaction
mixtures with enzyme heated to 95 °C for 10 min showed no
glucosyltransferase activity (data not shown).
Phenolic Extraction. Seven-day old cells were harvested by vacuum
filtration and freeze-dried. Phenolics were extracted from lyophilized
cells using 50 µL of a 97% methanol/3% formic acid solution per
milligram of cells. Cells were shaken and allowed to extract at 4 °C
overnight. Insoluble cell material was removed by centrifugation at
Glucosyltransferase activity toward resveratrol was strongly
pH dependent, with the maximal activity at pH 8.5-9 (Figure
3
). The Kms of the enzyme for its two substrates, resveratrol
and UDP-glucose, were 0.06 mM and 1.2 mM, respectively, at
pH 8.5 (Figure 4).
1
2 000g for 10 min. The supernatant was filtered through a 0.45 µM
pore filter prior to HPLC analysis.
Glucosyltransferase Activities and Phenolic Accumulation
Under Different Environmental Conditions. The concentra-
tion of piceid was highest in the dark grown cells. Visible light
treatment reduced piceid accumulation. Light + UVC further
decreased piceid concentration but strongly stimulated ac-
cumulation of resveratrol and another suspected stilbenoid in
these cells (Figure 5). Correspondingly, resveratrol glucosyl-
transferase activity was highest in extracts of the dark grown
cells and lower in extracts of cells grown under light and light
HPLC Analysis. The method used was adapted from Donovan et
al. (23) for separation of phenolics from fruits and juices. This method
was able to differentiate piceid (retention time ) 50 min) from
resveratrol (retention time ) 63 min).
Resveratrol Glucosylation Enzyme Assay. The method used was
a modification of the UFGT enzyme assay of Lister et al. (24). The
standard assay mixture consisted of 1.2 mM resveratrol (Sigma
Chemical Co., St. Louis, MO), 50 mM tricine buffer (pH 8.5), 5 mM
UDP-glucose, 6 mM dithiothrietol, and 50 µL of extract in a total
volume of 250 µL. Samples were incubated at 30 °C for 3 h; then the
reaction was stopped by the addition of 100 µL of 35% trichloroacetic
acid. Products were extracted with ethyl acetate. The ethyl acetate was
evaporated under vacuum, and the products were redissolved in 100
µL of methanol prior to HPLC analysis.
+
UV exposure (Table 1).
HPLC analysis of phenolic extracts of cells grown under
different light treatments showed a clear increase in anthocyanin
accumulation by visible light, with a lesser additional increase
upon UVC exposure (Figure 5). UFGT activity in extracts was
assayed using quercetin (a flavonol) as a substrate. Ford et al.
(25) showed that purified UFGT accepts quercetin as a substrate;
quercetin is preferred, because anthocyanidins are unstable at
â-Glucosidase Digestion. A 50-µL aliquot of cell extract or 60 µL
of resveratrol glucosylation reaction product were incubated with 1 unit
of â-glucosidase (Worthington Enzymes) in 0.1 M citrate buffer at pH
5
(total volume 500 µL). Digestion was carried out overnight at room

Polyphenol Glucosylating Activity in Grapes
J. Agric. Food Chem., Vol. 52, No. 11, 2004 3469
To investigate whether the observed changes in the concen-
trations of resveratrol and piceid were caused by direct
photochemical reactions, cell extracts (containing primarily
trans-piceid) and resveratrol (trans-form) were irradiated with
visible light. Trans-resveratrol isomerizes in visible light to the
cis form (12). Irradiated resveratrol showed a decrease in the
peak at 63 min (trans form), with a concomitant increase in the
peak at 68 min (cis form), which was absent when the sample
was incubated in the dark (Figure 6, parts A and B). Because
visible light isomerizes trans-resveratrol to the cis form, the
observed increase of trans-resveratrol in light and light + UV
treated cells could not have come from direct photochemical
isomerization. Piceid showed no such effect when irradiated
(
Figure 6, parts C and D). The addition of the glucose moiety
Figure 3. Resveratrol glucosylation pH optimum. Sodium phosphate (0.05
M) was used for pH 5, 6, and 7. Tricine (0.05 M) was used for pH 7.5,
prevents the light induced isomerization. In light of this finding,
the observed decrease of piceid also cannot be accounted for
by photochemical isomerization of trans-piceid.
8, 8.5, 9, and 10. All assays contained 1.2 mM resveratrol and 5 mM
UDP-glucose. The same enzyme preparation was used for all pH analyses.
No activity was observed at any pH without enzyme addition. All assays
were done in duplicate, except pH 8.5 and 9, which were done in
quadruplicate. Y-axis is mAU at 316 nm.
Is There More Than One Phenolic Glucosyltransferase?
It is possible that the observed activity of this enzyme is due to
the UFGT enzyme using resveratrol as a substrate. The observed
pH maxima of the enzyme activity is in agreement with those
of other glucosyltransferase enzymes (26-28). The Vitis Vinfera
UFGT enzyme has maximal activity at pH 8 (25). The Kms
for substrates (both UDPG and flavonoid/stilbenoid) are in
agreement with the calculated value for Vitis Vinifera UFGT
enzyme (25,28). There was glucosyltransferase activity toward
quercetin in these cells, though no glycosylated flavonols are
accumulated. It is possible that, in planta, which phenolic
glucosides accumulate is determined by which substrates are
provided to this multifunctional glucosyltransferase enzyme, and
the provision of substrates is controlled either by altering activity
of earlier biosynthetic enzymes, or by compartmentalization of
enzymes, substrates, or enzyme products. Specifically, the
accumulation of piceid in these cultures might occur because a
very active stilbene synthase enzyme provides resveratrol
substrate to a multifunctional glucosyltransferase enzyme
capable of utilizing both anthocyanidins and stilbenoids, both
planar molecules, as substrate.
Alternatively, in grape cells, there may be several glucosyltrans-
ferase enzymes, each of which is more specific toward one class
of phenolic substrates. In grapes, UFGT transcript accumulates
just as anthocyanins begin to accumulate at verasion (29). Enzy-
matic studies by Ford et al. (25) with this purified enzyme show
that it accepted several flavonoid substrates, but anthocyanidins
were acted upon more than 40-fold faster than any other. They
also found no activity using resveratrol as a substrate.
Figure 4. A Dependence of the reaction on UDP-Glucose concentration.
The resveratrol concentration was kept constant at 1.2 mM. B Dependence
of the reaction on resveratrol concentration. The UDP-glucose concentra-
tion was kept constant at 5 mM. All assays were done in triplicate. Insets
are Lineweaver−Burk plots of data (Y-axis ) 1/rate, X-axis ) 1/mM
substrate).
Evidence from other studies points to the existence of several
differentially regulated glucosyltransferase activities in Vitis.
Flavonol glucosides accumulate in response to sunlight after
anthocyanin accumulation had already begun (30). Piceid
accumulation can be strongly induced by methyl jasmonate
treatment in leaves and grapes both pre- and post-verasion (4).
Enzymatic analysis of crude grape cell extracts showed six
separable quercetin glycosylating activities, all showing lower
activity toward anthocyanidins as substrates (31). Grapes that
do not accumulate anthocyanins still accumulate other phenolic
glucosides.
the pH at which the glucosyltransferase assay was performed.
The glucosylating activity toward quercetin was similar from
the light and dark grown cells and was increased dramatically
by UVC treatment. This different responsiveness to light with
respect to quercetin and resveratrol glucosylation activity
suggests that there are at least two glucosyltransferase enzymes
active in the cultures. The rates of reactions for the different
substrates under different light conditions were also different.
Quercetin glycosylation occurred much more quickly, with rates
ranging between 82 and 172 nkatal/mg protein. Rates for
resveratrol glycosylation were much slower, between 15.2 and
Our results support the hypothesis that there is more than
one glucosyltransferase. The observation that a reduction in
resveratrol glucosylating activity correlated with reduced piceid
accumulation suggests that resveratrol glucosyltransferase activ-
ity directly determined the amount of piceid produced, but
resveratrol glucosylating activity had no correlation with the
anthocyanins accumulated. Dark-grown cells showed an increase
19.3 nkatal/mg protein (Table 1).

3470 J. Agric. Food Chem., Vol. 52, No. 11, 2004
Krasnow and Murphy
Figure 5. The effect of different light treatments of cultured cells on three stilbenoids and two anthocyanins extracted from the cells. Bars are the mean
of triplicate samples ± SE. Statistical analysis was a t-test at p ) 0.01 level. Y-axis is mAU at 316 nm for stilbenes on left graph and is mAU at 520
nm for anthocyanins on right graph. Black bars are dark grown cells, white bars are light grown cells, and hatched bars are light + UVC treated cells.
Table 1. Rates of Glucosylation of Resveratrol and Quercetin Under
Different Light Treatments by Cell Enzyme Extracts
glucosylation of
resveratrol
glucosylation of
quercetin
treatment
b
82.40 ± 4.73c
dark
light
light + UVC
19.37 ± 3.31
b
c
15.23 ± 3.04
90.54 ± 3.33
b
172.06 ± 10.51d
15.66 ± 1.37
a
All units are nkatal/mg total protein. Results are averages of triplicate samples
b
±
standard error. −dValues in a column followed by the same letter did not differ
by t-test at the p ) 0.01 level of confidence.
in resveratrol glucosylating activity and piceid accumulation,
relative to light-grown cells, but a strong decrease in anthocyanin
glucosides that were accumulated, suggesting a decrease in
UFGT activity using anthocyanidins as substrates. Activity
toward quercetin as a substrate had a different response to the
light treatments, with UV strongly stimulating glucosyltrans-
ferase activity toward this flavonol. The relative rates of
glucosylation with respect to stilbenes versus quercetin were
also markedly different.
On the basis of their responsiveness to environmental stimuli,
there is evidence of three distinct glucosyltransferase activities
in our cell extract. The activity of resveratrol glucosylation
decreased upon visible light irradiation of the cultures. This
correlated with the observed decrease in accumulation of piceid
in these cells. A quercetin glucosylating enzyme was stimulated
only when irradiated with UV light, with similar activity in dark
grown or light grown cells. The observed increase in antho-
cyanin accumulation in light irradiated cultures suggested an
increase in the activity of an anthocyanidin utilizing glucosyl-
transferase upon light or light + UV treatment.
Figure 6. The effect of visible light irradiation of trans-resveratrol and
cell extract containing trans-piceid. All irradiations were carried out at 12 °C
using a tungsten halogen lamp (1300 µmol photons/m -s). All chropmato-
graphs were monitored for absorbance at 316 nm. A is trans resveratrol
incubated in the dark B is trans resveratrol incubated overnight under
constant irradiation. Retention time for trans-resveratrol is 63 min, for cis-
resveratrol is 68 min. C is cell extract incubated in the dark. D is cell
extract irradiated overnight. Peaks at 55 and 59 min in cell extracts are
due to absorbance by anthocyanins acylated with hydroxycinnamic acids.
2
in the phenolic pathway found only as a single band (32). It is
possible that the heterologous probe used by this group failed
to recognize other glucosyltransferase genes. Alternatively, there
might be a single gene that can be differentially spliced
producing proteins with different substrate specificities.
Plants must deal with an ever-changing environment and with
their own developmental changes. Being immobile, many of
Several glucosyltransferase genes, all producing enzymes
capable of glycosylating phenolic substrates would be expected
to have homology to one another. In Southern blot analyses of
Vitis Vinifera genomic DNA, UFGT was one of the few enzymes

Polyphenol Glucosylating Activity in Grapes
J. Agric. Food Chem., Vol. 52, No. 11, 2004 3471
these alterations take the form of the synthesis of specific
chemical compounds. Grapes are capable of synthesizing many
distinct polyphenolic glucosides, including piceid, flavonol
glucosides, and anthocyanins. Each of these compounds pre-
sumably has a function in the plant. Anthocyanins attract animals
to distribute the seeds of mature red fruit (33). It is important
that biosynthesis of these pigments be controlled developmen-
tally, so that pigment accumulation does not begin until the seeds
are mature and ready to be dispersed. Flavonol glucosides have
been shown to be synthesized in the dermal tissues of many
plant genera in response to UV light; the flavonols absorb
strongly in the UV wavelengths, protecting underlying tissues
from UV-induced damage (34, 35). It is unclear why grape cells
accumulate piceid, although it is more water soluble than
resveratrol and the glucosylation prevents photochemical isomer-
ization of resveratrol moiety. Possibly a better reason to
accumulate the glucoside is that the glucosylation protects
resveratrol from oxidation by fungal tyrosinase (36). Assuming
that these glucosides have specific roles for the plant at specific
developmental stages or environmental conditions, it is important
for the plant to stringently regulate the glucosylation of
polyphenolic compounds. Having several specific glucosyl-
transferases that are responsive to different environmental cues
allows for the fine-tuning of the chemical compounds produced
by the plant to best suit its growing conditions.
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We have shown, for the first time, that there is a glucosyltrans-
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1
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