Formation of UV-honey guides in Rudbeckia hirta

Article abstract of DOI:10.1016/j.phytochem.2009.04.017

The UV-honey guides of Rudbeckia hirta were investigated by UV-photography, reflectance spectroscopy, LC-MS analysis and studies of the enzymes involved in the formation of the UV-absorbing flavonols present in the petals. It was shown for the first time

Full text of DOI:10.1016/j.phytochem.2009.04.017

Phytochemistry 70 (2009) 889–898
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Formation of UV-honey guides in Rudbeckia hirta
a
a
a
a
b
c
Karin Schlangen , Silvija Miosic , Ana Castro , Karin Freudmann , Maria Luczkiewicz , Florian Vitzthum ,
c
d
e
a,
*
Wilfried Schwab , Sonja Gamsjäger , Maurizio Musso , Heidi Halbwirth
a
Technical University of Vienna, Institute for Chemical Engineering, Getreidemarkt 9/1665, A-1060 Vienna, Austria
Akademia Medyczna w Gda n´ sku, Katedra i Zakład Farmakognozji, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
Technical University of Munich, Biomolecular Food Technology, Lise-Meitner-Str. 34, 85354 Freising, Germany
Ludwig Boltzmann Institute of Osteology, The Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Medical Department,
Heinrich Collin-Str. 30, A-1140 Vienna, Austria
b
c
d
e
University of Salzburg, Department of Materials Research and Physics, Hellbrunnerstrasse 34, 5020 Salzburg, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
The UV-honey guides of Rudbeckia hirta were investigated by UV-photography, reflectance spectroscopy,
LC–MS analysis and studies of the enzymes involved in the formation of the UV-absorbing flavonols pres-
ent in the petals. It was shown for the first time that the typical bull’s eye pattern is already established at
the early stages of flower anthesis on the front side of the petal surface, but is hidden to pollinators until
the buds are open and the petals are unfolded. The rear side of the petals remains UV-reflecting during
the whole flower anthesis. Studies on the local distribution of 19 flavonols across the petals confirmed
that the majority are concentrated in the basal part of the ray flower. However, in contrast to the earlier
studies, eupatolitin 3-O-glucoside (6,7-dimethoxyquercetin 3-O-glucoside) was present in both the basal
and apical parts of the petals, whereas eupatolin (6,7-dimethoxyquercetin 3-O-rhamnoside) was exclu-
sively found in the apical parts. The enzymes involved in the formation of the flavonols in R. hirta were
demonstrated for the first time. These include a rare flavonol 6-hydroxylase, which was identified as
cytochrome P450-dependent monooxygenase and did not accept any methylated flavonol as substrate.
All enzymes were present in the basal and apical parts of the petals, although some of them clearly
showed higher activities in the basal part. This indicates that the local accumulation of flavonols in R. hir-
ta is not achieved by a locally restricted presence of the enzymes involved in flavonol formation.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 October 2008
Received in revised form 22 April 2009
Available online 26 May 2009
Keywords:
Rudbeckia hirta
Asteraceae
UV-honey guides
Flavonoids
Methylated flavonols
A-ring-specific hydroxylation
Flavonoid 6-hydroxylase
Methyltransferases
Glucosyltransferases
1
. Introduction
Honey guides are part of the pigmentation pattern, which oc-
absorbing substances and are therefore invisible to the human
eye. Such UV-patterns on flower petals may be perceived by many
pollinating insects because of their specific color sense, which
makes them blind to scarlet red coloration but sensitive to the
UV-range of the spectrum (Eisner et al., 1969; Silberglied, 1979;
McCrea and Levy, 1983; Dyer, 1996; Indsto et al., 2006, 2007).
The prime example for the formation of UV-honey guides is
Rudbeckia hirta (Fig. 1). In 1972, Thompson et al. were able to show
that the flowers, which are uniformly yellow to the human eye,
have a characteristic UV-absorbing centre, whilst the outer parts
of the ray petals are UV-reflecting. For UV-sensitive insects, the
Rudbeckia flower appears as a bull’s eye with two concentric
dichromatic circles against the green meadow background (Abra-
hamson and McCrea, 1977). The UV-reflecting apical part is
responsible for the long-distance orientation and acts as a landing
site for the pollinators, whereas the UV-absorbing centre of the
flower acts as a ‘honey guide’, helping the pollinators to orient
themselves within the flower after landing (Daumer, 1956, 1958;
Jokl and Fürnkranz, 1989; Burr et al., 1995). In contrast to the fully
developed Rudbeckia flower, small buds were described as being
curs particularly frequently in bee-pollinated flowers. Their physi-
ological function is to guide pollinators to the reproduction organs
and to the nectar in the flower centre (Daumer, 1956, 1958; Kevan
and Mulligan, 1973; Harborne, 1993; Kevan et al., 2001). A broad
variety of forms are known. They may appear as color contrast –
as in snapdragons (Antirrhinum majus) where a yellow spot is lo-
cated on the lip of an otherwise red or orange flower – or may take
the form of colored dots or lines in particular areas of the petals.
Often, honey guides are formed by local accumulation of UV-
Abbreviations: CHI, chalcone isomerase; CHS, chalcone synthase; DHK,
dihydrokaempferol; EGME, ethylene glycol mono methyl ether; EtOAC, ethyl
0
0
acetate; FHT, flavanone 3-hydroxylase; FLS, flavonol synthase; F3 H, flavonoid 3 -
hydroxylase; F6H, flavonol 6-hydroxylase; F7GT, flavonol 7-O-glucosyltransferase;
F3GT, flavonol 3-O-glucosyltransferase; Glu, glucose; Rha, rhamnose; SAM, S-
Adenosylmethionine; UDPG, UDP-glucose.
*
Corresponding author. Tel.: +43 1 58801 17311; fax: +43 1 58801 17399.
E-mail address: hhalb@mail.zserv.tuwien.ac.at (H. Halbwirth).
0
031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.04.017

8
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K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
uniformly colored to the insect’s eyes. This was explained as part of
the ontogenetic development, because only fertile flowers rely on
pollinator attraction (Jokl and Fürnkranz, 1989; Burr et al., 1995).
First chemical analysis revealed that local accumulation of three
flavonols at the petal base is responsible for the basal UV-absorb-
ing zones of the mature ray flower (dark in Fig. 1): quercetagetin
et al., 1973; Jokl et al., 1999; Dyer et al., 2004; Chittka and Kevan,
2005). In contrast to the earlier studies, which commonly show
UV-photos of whole plants or flowers (Jokl and Fürnkranz, 1989;
Burr et al., 1995), we photographed the front- and rear sides of sin-
gle petals of four stages of flower anthesis with UV-transmittable
filters. Even the youngest petals exhibited the typical bull’s eye pat-
tern at their surface (Fig. 3A). In contrast, the rear sides of the petals
remain UV-reflecting in all developmental stages (Fig. 3B). Thus, the
honey guides are already formed at early stages but are hidden to
the pollinators, because juvenile Rudbeckia petals are two times
folded and only the rear side is visible at the early stages of flower
anthesis. Apparently, the bull’s eye is formed only on the front side
of the flower as the relevant side for pollen and nectar collection.
UV-photography (Williams and Williams, 1993) gives a qualita-
tive picture of the reflectance distribution across the petal surface
for a selected, rather narrow wavelength range (in this case around
360 nm ± 20 nm) and also runs the risk of interpretational difficul-
ties (Kevan, 1979). In contrast, reflectance spectroscopy can pro-
vide a quantitative value of spectral reflectance for selected areas
of the petal over a wide spectral range from UV to near infrared
(Galsterer et al., 1999; Langanger et al., 2000). In the present study,
reflectance curves (Fig. 4, Table 1) of both petal sides were also ob-
tained. Each reflectance curve is the average value obtained from
five different petals of the same flower, measured either at the api-
cal or at the basal area of the petal. Additionally shown in Fig. 4 is
the standard deviation of the mean value, given as 95% confidence
interval.
(
6-hydroxyquercetin), patulitrin (6-methoxyquercetin 7-O-gluco-
side) and 6,7-dimethoxyquercetin 3-O-glucoside (Thompson
et al., 1972) (Fig. 2). It is remarkable that all the three flavonols
show an extra hydroxyl or methoxyl group in position 6 in addition
to the basic 5,7-hydroxylation pattern of the common flavonols,
which considerably influences the light absorption. Apart from
their UV-absorbance, they also contribute to the yellow color of
the flower. These so-called ‘yellow flavonols’ are particularly
formed in members of Asteraceae and Leguminosae (Harborne,
1
967, 1976).
Interestingly, carotenoids are also present in R. hirta. In contrast
to the flavonols, they are uniformly distributed in the flowers
Thompson et al., 1972). The co-occurrence of two yellow pigment
(
types in flowers has frequently been observed, particularly in
highly evolved plant species (Harborne, 1988). At the first sight,
this seems to be a waste of biochemical resources, which could
be justified only by their different physiological functions. Actually,
the co-occurrence of different yellow pigment types is frequently
connected to the formation of UV-honey guides (Harborne,
1
988). In contrast to the UV-absorbing flavonols in the inner part,
UV-reflecting carotenoids are responsible for the formation of the
bright circle of the ray disk of R. hirta.
In addition to the flavonols identified by Thompson et al. (1972),
further flavonols were isolated later from R. hirta petals (Cisowski
et al., 1993): quercimeritrin (quercetin 7-O-glucoside), quercetagi-
trin (quercetagetin 7-O-glucoside), 6,7-dimethoxyquercetin and
Whereas the rear side of the flower petal does not show any sig-
nificant difference between the reflectance of the apical and the
basal part of the petal during anthesis over the whole spectral
range, for the front side this difference is already perceptible in
the ultraviolet spectral range in anthesis stages ST1 and ST2, and
becomes quite significant during the anthesis stages ST3 and ST4.
6
,7-dimethoxyquercetin 3-O-rhamnoside (Fig. 2). However, their
contribution to the formation of UV-honey guides has not been
studied so far. We investigated the formation of UV-honey guides
in R. hirta by UV-photography, reflectance spectroscopy, LC–MS
analysis and biochemical studies. We provide an in-depth insight
into the local distribution of different flavonols in R. hirta petals
and show for the first time the biochemical pathway leading to
the pigments involved in the formation of UV-honey guides.
2.2. Local distribution of flavonols across the Rudbeckia petals
We investigated the local distribution of 19 flavonols in basal
and apical parts of the petals in four different anthesis stages. The
studies comprised the compounds previously described in R. hirta
(Thompson et al., 1972; Cisowski et al., 1993) and the related flavo-
nols, which could be potential intermediates or end-products in the
flavonol formation in R. hirta. This included kaempferol (Km),
kaempferol 3-O-glucoside (Km3glu), kaempferol 7-O-glucoside
2
. Results and discussion
(
Km7glu), quercetin (Qu), quercetin 3-O-glucoside (Qu3glu), quer-
2.1. UV-photography and UV-reflectance spectroscopy
cetin7-O-glucoside (Qu7glu), 6-hydroxykaempferol (6-OHKm),
quercetagetin (Quag), quercetagetin 3-O-glucoside (Quag3glu),
quercetagetin 7-O-glucoside (Quag7glu), 6-methoxykaempferol
UV-photography is a helpful and well-established tool for visu-
alizing floral patterns that are invisible to the human eye (Kevan
(
6-MeOKm), 6,7-dimethoxykaempferol 3-O-rhamnoside (6-MeO-
Km3rha), 7,8-dimethoxykaempferol 3-O-glucoside (7,8-DiM-
eOKm3glu), patuletin (6-MeOQu), patulitrin (6-MeOQu7glu),
eupatolitin (6,7-DiMeOQu), eupatolitin 3-O-glucoside (6,7-DiM-
eOQu3glu), eupatolin (6,7-DiMeOQu3rha), and eupatolitin 3-O-
rhamnoglucoside (6,7-DiMeOQu3glu-rha) (Fig. 2).
Qu7glu, Quag7glu, 6-MeOQu7glu, 6,7-DiMeOQu3glu and 6,7-
DiMeOQu3rha were the main flavonols present in the R. hirta
petals. Km3glu, Qu3glu, 6-MeOQu, and 6,7-DiMeOKm3rha were
present in medium amounts and only small amounts of Qu, Quag
and 6,7-DiMeOQu3glu-rha could be detected. Km, Km7glu,
6
-OHKm, 6-MeOKm, and 7,8-DiMeOKm3glu could not be detected
in the petals. The LC–MS spectra indicated the presence of further
unidentified flavonols (data not shown), which were not included
in the current study.
During the flower development, flavonol concentration in-
creased continuously. The majority of the flavonols present in the
petals were concentrated at the basal parts of the petals. Fig. 5
shows the local distribution of the main flavonols across the R. hir-
Fig. 1. UV-photography of R.hirta cv. ‘Indian Summer’, showing the reflectance at
around 360 nm. The circular dark honey guide is known as ‘bull’s eye-effect’.

K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
891
R4
OH
R3
B
R2
R1
O
C
A
R
OH
O
Name
Abbreviation
Km
R
R
H
H
H
H
H
H
H
H
1
R
2
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
R
H
H
H
H
4
Kaempferol
OH
OGlu
OH
OH
OH
OGlu
OH
OH
OH
OH
OH
Kaempferol 3-O-glucoside
Kaempferol 7-O-glucoside
Km3glu
OH
Km7glu
OGlu
7
-Methoxykaempferol
7-MeOKm
Qu
OCH
OH
3
Quercetin
OH
OH
OH
OH
H
Quercetin 3-O-glucoside
Quercetin 7-O-glucoside
Qu3glu
OH
Qu7glu
OGlu
7
-Methoxyquercetin
-Hydroxykaempferol
7-MeOQu
6-OHKm
Quag
OCH
OH
3
6
OH
OH
Quercetagetin
OH
OH
OH
OH
H
Quercetagetin 3-O-glucoside
Quercetagetin 7-O-glucoside
Quag3glu
Quag7glu
6-MeOKm
6-MeOQu
6,7-DiMeOQu
6-MeO Qu7glu
7,8-DiMeOKm3glu
OGlu OH
OH
OH
OH
OH
OH
OH
OH
OGlu
OH
6
-Methoxykaempferol
OCH
OCH
OCH
OCH
3
3
3
3
3
3
Patuletin (6-methoxyquercetin)
Eupatolitin (6,7-dimethoxyquercetin)
Patulitrin (patuletin 7-O-glucoside)
OH
OH
OH
OH
H
OCH
OGlu
OH
3
7
6
,8-Dimethoxykaempferol 3-O-glucoside
OGlu OCH
ORha OCH
OCH
H
3
,7-Dimethoxykaempferol 3-O-rhamnoside 6,7-DiMeOKm3rha
OCH
3
H
Eupatolitin 3-O-glucoside
6,7-DiMeOQu3glu
6,7-DiMeOQu3rha
OGlu OCH
ORha OCH
3
3
OCH
OCH
3
3
H
H
OH
OH
Eupatolin (Eupatolitin 3-O-rhamnoside)
Fig. 2. Chemical structures of flavonols included in the studies.
ta petal (black columns: basal parts, white columns: apical parts).
However, in contrast to the earlier studies, considerable amounts
of 6,7-DiMeOQu3glu could be detected in both petal parts and
synthase (CHS), chalcone isomerase (CHI) and flavanone 3-hydrox-
ylase (FHT) (Forkmann and Heller, 1999). The presence of all en-
zymes involved in flavonol formation could be demonstrated in
preparations from R. hirta (Fig. 6). In addition, a high flavonoid
6
,7-DiMeOQu3rha and 6,7-DiMeOKm3rha were present exclu-
0
sively in the apical parts (Fig. 5).
3 -hydroxylase (F3’H) activity could be detected which correlates
well to the fact that the majority of flavonols in R. hirta have a
0
0
2.3. Biochemical pathway leading to the flavonols present in R. hirta
3 ,4 -hydroxylation pattern in ring B.
The spectrum of flavonols present in R. hirta indicated the pres-
ence of a number of modifying enzymes which catalyzed the (i)
introduction of a hydroxyl group in position 6, (ii) methylation of
hydroxyl groups in positions 6 and 7 and (iii) glucosylation of hy-
droxyl groups in positions 3 and 7.
Flavonol biosynthesis is well established and has been studied
in many different plants (Forkmann and Heller, 1999). The key step
in the flavonol formation is the introduction of a double bond be-
tween C-2 and C-3 of dihydroflavonols, which is catalyzed by flavo-
nol synthase (FLS), a 2-oxoglutarate-dependent dioxygenase. The
dihydroflavonols are formed by the consecutive action of chalcone
Up to now, hydroxylation in position 6 has been observed only
in a few plant species (Anzellotti and Ibrahim, 2000, 2004; Latun-

8
92
K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
Fig. 3. UV-photography of four developmental stages (from left to right: ST1, ST2, ST3, and ST4) of Rudbeckia petals, showing the reflectance at around 360 nm. A: front side;
B: rear side.
0
de-Dada et al., 2001; Halbwirth et al., 2004). Interestingly, two dif-
ferent enzyme systems were shown to catalyze this reaction in the
flavonoid pathway. Chrysosplenium americanum, a semiaquatic
weed, accumulates a variety of partially methylated flavonol gluco-
sides in the leaves. In this plant, hydroxylation in position 6 is cat-
flavones, dihydroflavonols and 6 -deoxychalcones were not ac-
cepted as substrates either.
The enzymatic formation of Quag was studied in more detail
(Table 2). The reaction was strictly dependent on NADPH and oxy-
gen, and the activity was significantly reduced when NADPH was
substituted by NADH (Table 2). Highest reaction rates were ob-
served in the presence of 1.45 mM NADPH, at higher concentra-
tions, the F6H activity decreased continuously. Addition of FAD
resulted in slightly increased enzyme activities (Table 2). Highest
reaction rates were measured at pH 7.0 and at 30 °C, where the for-
mation of Quag was linear with time up to 15 min and with protein
alyzed by
a
2-oxoglutarate-dependent dioxygenase, which
requires methylated flavonols as substrates (Anzellotti and Ibra-
him, 2000, 2004). Five distinct O-methyltransferases, which have
high specificity for a definite position, sequentially methylate the
hydroxyl groups of the flavonol substrates. It was shown that
methylation in positions 3 and 7, but not necessarily in position
0
4
, precedes the hydroxylation in position 6 in C. americanum (Ibra-
concentration up to 8 lg protein in the standard assay. At 10 °C,
him et al., 1987).
the rate was about 50% of the maximum; at temperatures higher
than 30 °C the reaction rate decreased strongly. The highest spe-
cific activities were measured in the developmental stage 2, where
the specific activity was 222 nkat/mg protein under standard con-
ditions. The cytochrome P450-specific inhibitors ketoconazole and
tetcyclacis clearly reduced enzyme activity, whereas ancymidol,
had only a weak influence (Table 2). The addition of a crude frac-
tion of cytochrome P450 reductase-specific antibodies led to a total
loss of enzyme activity, whereas heat-inactivated antibodies did
not have any effect (Table 2).
The localization in the microsomal fraction, the dependence on
NADPH and oxygen and the specific inhibition by cytochrome P450
reductase antibodies (Benveniste et al., 1989; Nielsen and Møller,
1999; Halbwirth et al., 2004) clearly indicate that the F6H of R. hir-
ta is a cytochrome P450-dependent enzyme. The introduction of an
additional hydroxyl group in ring A is the first modification step in
the biosynthetic pathway to the flavonols formed in R. hirta, be-
cause methylated and/or glucosylated flavonols are not accepted
as substrates. In addition, the formation of flavonols has to precede
A-ring hydroxylation because flavanones and dihydroflavonols are
not hydroxylated in position 6. Thus, it is obvious that the meth-
oxylated flavonols present in R. hirta are formed in a different
way than those in C. americanum.
In contrast, a cytochrome P450-dependent monooxygenase cat-
alyzes the formation of Quag from Qu in Tagetes patula, but does
not convert any partially methylated flavonols (Halbwirth et al.,
2
004). In T. patula, glycosylated 6-hydroxyflavonols contribute to
the yellow color of the flower (Tarpo, 1969).
As C. americanum, R. hirta contains partially methylated flavo-
nols, but the pigments described so far show methoxy groups only
in positions 6 and 7. We investigated whether there was a defined
sequence for the hydroxylation, methylation and glycosylation of
the flavonols in R. hirta and which enzyme class is responsible
for the introduction of the additional group in position 6. From
the substrate specificities and conversion rates observed, the po-
tential pathway leading to the flavonols identified from R. hirta
was suggested (Fig. 7).
2
.3.1. Hydroxylation in position 6
Incubation of [14C]Qu with crude enzyme preparations from
petals of R. hirta in the presence of NADPH led to the formation
1
4
of [ C]Quag, which was identified as previously described by HPLC
co-chromatography with the authentic reference substance using a
photodiode array detector coupled with a radioactivity detector
(Halbwirth et al., 2004, 2006). No product formation was observed
in the presence of typical dioxygenase cofactors (2-oxoglutarate,
2
+
ascorbate, and Fe ). Preparations of the microsomal protein frac-
tion showed a 3.5-fold enrichment of the enzyme activity com-
pared to the crude extract, and only 8% of the activity remained
in the supernatant after the final centrifugation step, thus indicat-
ing that the enzyme is membrane bound. Therefore all further
enzymatic studies were performed with the microsomal fraction.
When Km was used as a substrate, the formation of Quag, but
2.3.2. Methylation of hydroxyl groups of flavonols
Incubation of Qu with enzyme preparations from petals of R.
1
4
hirta in the presence of [ C]S-Adenosylmethionine (SAM) led to
1
4
the formation of [ C]7-MeOQu, which was identified by HPLC
co-chromatography with the authentic reference substance using
a photodiode array detector coupled with a radioactivity detector.
When Km was used as a substrate, 7-MeOKm was formed
accordingly. Incubation of Quag with crude enzyme preparations
0
not of 6-OHKm, was observed, which is a result of the high F3 H
1
4
activity present in the microsomal fraction. No reaction products
were detected, when Quag3glu, Quag7glu or methylated quercetin
derivatives were incubated under the same conditions. Flavanones,
from petals of R. hirta in the presence of [ C]SAM primarily
resulted in the formation of 6-MeOQu. In addition, a by-product
was formed in very low amounts which was identified as 6,7-DiM-

K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
893
wavelength [nm]
wavelength [nm]
300
400
500
600
700 300
400
500
600
700
8
6
4
2
0
0
0
0
apex mean
ST1 front
apex mean
ST1 rear
80
60
40
20
apex confidence 95%
base mean
apex confidence 95%
base mean
base confidence 95%
base confidence 95%
0
0
8
0
0
0
0
apex mean
ST2 front
apex mean
ST2 rear
80
apex confidence 95%
base mean
apex confidence 95%
base mean
base confidence 95%
base confidence 95%
6
4
2
60
40
20
0
0
0
ST3 front
8
6
4
2
apex mean
apex mean
ST3 rear
80
apex confidence 95%
base mean
apex confidence 95%
base mean
base confidence 95%
base confidence 95%
0
0
0
60
40
20
0
0
8
0
0
0
0
apex mean
ST4 front
apex mean
ST4 rear
80
apex confidence 95%
base mean
apex confidence 95%
base mean
base confidence 95%
base confidence 95%
6
4
2
60
40
20
0
0
300
400
500
600
700 300
400
500
600
700
wavelength / nm
wavelength [nm]
Fig. 4. Reflectance spectra obtained from the apical part and the basal part of the front side and the rear side of R. hirta petals during flower anthesis (compare Fig. 3 with the
UV-photography of the four developmental stages ST1, ST2, ST3, ST4). Each reflectance curve is the average value obtained from five different petals of the same flower;
additionally shown is the standard deviation of the mean value, given as 95% confidence interval.
eOQu. Glucosylated flavonols were accepted as substrates only to a
very low extent.
the apical part. However, the other three anthesis stages did not
show these pronounced differences of the methyltransferase
(MT) activities.
The formation of 7-MeOQu showed a maximum at pH 7.5,
whilst formation of 6-MeOQu and 6,7-DiMeOQu slightly increased
at higher pH values (with a maximum at pH 8.5). Using Quag as a
substrate, highest reaction rates were measured at 30 °C, where
the formation of 6-MeOQu was linear with time up to 15 min
2.3.3. Glucosylation of hydroxyl groups of flavonols
Incubation of Quag with enzyme preparations from petals of
1
4
R. hirta in the presence of [ C]UDP-glucose (UDPG) led to the
1
4
and with protein concentration up to 50
assay. However, at 25 and 40 °C, 75% of the maximum activity at
0 °C was still observed, whereas at higher temperatures, the con-
l
g protein in the standard
formation of [ C]Quag7glu, which was identified by HPLC co-
chromatography with the authentic reference substance using a
photodiode array detector coupled with a radioactivity detector.
3
14
version rates decreased drastically. The highest specific activities
were measured in the developmental stage 4, where the activity
in the basal part of the petals was two times higher than that in
When Qu was used as a substrate, the formation of [ C]Qu7glu
1
4
and [ C]Qu3glu was observed. We assume that two different
glucosyltransferases, flavonol 7-O-glucosyltransferase (F7GT)

8
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K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
Qu7glu
Quag7glu
6-MeOQu7glu
1
00
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
1
2
3
4
1
2
3
4
1
2
3
4
Anthesis stage
Anthesis stage
Anthesis stage
6,7-DiMeOQu
6,7-DiMeOQu3gl
6,7-DiMeOQu3rha
1
00
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
1
2
3
4
1
2
3
4
1
2
3
4
Anthesis stage
Anthesis stage
Anthesis stage
6-MeOQu
Km3glu
6,7-DiMeOKm3rha
1
00
1
00
1
00
80
60
40
20
0
8
6
4
0
0
0
8
0
0
0
0
0
6
4
2
20
0
1
2
3
4
1
2
3
4
1
2
3
4
Anthesis stage
Anthesis stage
Anthesis stage
Fig. 5. Relative distribution (% rel.) of selected flavonols in basal (black columns) and apical (white columns) parts of R. hirta petals in four anthesis stages. Values were
calculated in relation to the highest amount of each compound present.
and flavonol 3-O-glucosyltransferase (F3GT), are present in the
petals of R. hirta, because glucosyltransferases frequently act spe-
cifically on a distinct position (Stich et al., 1997; Forkmann and
Heller, 1999).
rates were measured at pH 7.5 and at 30 °C, where the formation
of 6,7-DiMeOQu3glu was linear with time up to 40 min and with
protein concentration up to 20 lg protein. The highest specific
activities were measured in the developmental stage 3. F3GT did
not show these pronounced activity differences between basal
and apical parts of the petals as observed for F7GT (Fig. 6).
Apart from Qu and Quag, Km, 6-OHKm, 6-MeOKm and 6-MeO-
Qu were also glucosylated in position 7. However, the highest
reaction rates were observed with Quag and 6-OHKm as sub-
strates followed by 6-MeOQu and 6-MeOKm. With Qu and Km
as substrates, 10-fold lower activities were observed. This indi-
cates that in the biochemical pathway, glucosylation tends to pre-
cede the methylation reaction. For all substrates tested, the
glucosylation in position 7 showed pH optima between pH 7.5
and 8.25 (Quag, 6-MeOKm: 7.75; Qu: 7.5; Km, 6-OHKm: 8.0; 6-
MeOQu: 8.25).
Using Quag as a substrate, the highest reaction rates were mea-
sured at 30 °C, where the formation of Quag7glu was linear with
time up to 25 min and with protein concentration up to 15 lg pro-
tein in the standard assay. The highest specific activities were mea-
sured in anthesis stage 4. Interestingly, F7GT activity was much
higher in the basal part than that in the apical part of the petals
2.4. Formation of UV-honey guides
UV-photographs of Rudbeckia flowers suggest the absence of
flavonols in the apical part of the petals and actually, several flavo-
nols could only be found in the basal parts (Thompson et al., 1972).
Thus we assumed a local restriction of the flavonol biosynthesis
and the absence of the involved enzyme in the apical parts. Our
investigations have confirmed that many flavonols are concen-
trated in the basal parts of the flowers (Table 1), whereas carote-
noids are present in the basal and apical parts of the petals in
the four developmental stages. However, in contrast to earlier
studies, 6,7-DiMeOQu3glu was surprisingly found in the basal
and apical parts and 6,7-DiMeOQu3rha was found exclusively in
the apical parts.
(Fig. 6), which correlates well with the fact that only traces of fla-
vonol 7-O-glucosides could be detected in the apical parts (Fig. 2).
Compared to the F7GT activity, glucosylation in position 3 was
distinctly lower. The highest reaction rates were observed using
The presence of two flavonol pigments in the apical part of the
ray flower raises the question of why this does not lead to UV-
absorption in the outer part. It could be speculated that the con-
centration gradient of flavonols in the different parts of the petals
is high enough to result in the differences in the UV-absorption ob-
served. In addition, the flavonol 7-O-glucosides are exclusively
accumulated in the basal parts. In contrast to the 3-O-glucosides,
they show yellow fluorescence in the UV-light and this could also
6
,7-DiMeOQu as a substrate. With Km and Qu as substrates, the
reaction rates were 10 times lower. No flavonol 3-O-glucosides
were formed, when Quag, 6-MeOQu, 6-OHKm or 6-MeOKm were
incubated with enzyme preparations of R. hirta in the presence of
UDPG. Using 6,7-DiMeOQu as a substrate, the highest reaction

K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
895
CHS
FHT
FLS
8
6
4
2
00
00
00
00
0
3000
3000
2000
1000
0
2
1
000
000
0
1
1
1
2
3
4
4
4
1
1
1
2
3
4
1
2
3
4
anthesis stage
anthesis stage
anthesis stage
F3'H
F6H
MT
3
2
1
000
000
000
0
300
200
100
0
3000
2000
1000
0
2
3
2
3
4
1
2
3
4
anthesis stage
anthesis stage
anthesis stage
F7GT
F3GT
1
000
80
60
5
00
40
20
0
0
2
3
2
3
4
anthesis stage
anthesis stage
Fig. 6. Specific activities (nkat/kg protein) of selected flavonoid enzymes in basal (black) and apical parts (white) of the petals in four anthesis stages. F7GT and MT assays
were performed with quercetagetin as substrate, F3GT with quercetin.
contribute to the pollinator’s perception of the flower as two con-
centric dichromatic circles. However, 3-O-glucosides and 7-O-glu-
cosides of flavonols may not be differentiated by UV-photography
because both show UV-absorption at 360 nm and therefore may
appear dark.
niques. Work on the identification of further flavonols present in
R. hirta is also in progress.
3
. Experimental
In accordance with the presence of 6,7-DiMeOQu3glu, the activ-
ities of all enzymes involved in its formation could be demon-
strated in both parts of the petals (Fig. 6). For FHT, FLS, F6H and
F7GT, the activities in the basal parts were considerably higher
than that in the apical parts. However, a strict ± system could
not be observed. The methylating and glucosylating enzymes
showed highest activities in the late developmental stages,
whereas most other enzymes had a climax in stage 2. There was
no indication that flavonol biosynthesis increases at the end of
the flower life-cycle. The absence of 6,7-DiMeOQu3rha in the basal
parts of the petals could be explained by a local lack of flavonol 3-
O-rhamnosyltransferase activity. However, up to now, we have not
been able to demonstrate the presence of this enzyme in any part
of the petal, despite continued efforts.
The previous work on anthesis in Asteraceae reported that the
bull’s eye pattern is present only in fertile flowers whilst juvenile
and senescent flowers appear uniformly dark UV-absorbing (Jokl
and Fürnkranz, 1989). Our investigations have shown that in R. hir-
ta the bull’s eye pattern is established already in the earliest devel-
opmental stages. However, as long as the buds are closed and the
petals are folded, the honey guides remain invisible to pollinators,
because the rear side is uniformly UV-reflecting. This implicates
that the honey guide forming pigments are accumulated only in
the surface cell layers of the petals. In this case, a potential ± sys-
tem would not be detected with enzyme preparations from the
whole basal and apical parts. Further work will concentrate on
the identification of the corresponding genes involved and the
detection of their local expression by in situ hybridization tech-
3
.1. General experimental procedures
UV photography was carried out with a Nikon FM2 camera with
a UV-Nikkor lens (f = 105 mm, 1:4.5, Nikon, Vienna, Austria) and a
T-Max 100 black & white film 135-36 (Kodak, Vienna, Austria)
(Williams and Williams, 1993). The system used for LC-UV-ESI-
MSn analysis was a Bruker esquire 3000 plus mass spectrometer,
equipped with an Agilent 1100 HPLC system (Agilent, Waldbronn,
Germany) composed of an Agilent 1100 quaternary pump and an
Agilent 1100 variable wavelength detector. HPLC analysis was per-
formed on a Perkin Elmer Series 200 system (Perkin Elmer, Vienna,
Austria) equipped with a photodiode array detector coupled with a
5
00TR Flow Scintillation Analyzer for the detection of radiolabelled
substances. Radiolabelled substances were also detected with a
TLC Linear Analyzer (Berthold LB 2842, Wildbad, Germany) or with
a Winspectral scintillation counter (Wallac, Vienna, Austria).
3.2. Plant material
The investigations were performed on R. hirta cv. ‘Indian Sum-
mer’ (Austrosaat, Vienna, Austria). The plant material was culti-
vated in the municipal parks of Vienna, collected during the
summer periods 2005 and 2006, shock frozen in liquid nitrogen
and stored at ꢀ80 °C. Morphological criteria were used for sub-
dividing the developmental process into four anthesis stages
(Fig. 3). ST1 and 2 refer to closed buds (5 and 10 mm petal length,
respectively), ST3 refers to buds just opening (petal length 15 mm)
and ST4 refers to open flowers (petal length 20 mm).

8
96
K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
CHS
OH
OH
OH
OH
HO
O
O
HO
O
O
F3’H
F6H
OH
Eriodictyol
Naringenin
FHT
FHT
OH
OH
OH
OH
OH
HO
O
HO
O
MeO
MeO
O
O
F6H
OH
F3’H
oH
OGlu
OH
O
OH
O
OH
Dihydrokaempferol
FLS
Dihydroquercetin
Eupatoletin 3glu
FLS
OH
F3GT
F3GT
F3GT
OH
OH
OH
OH
OH
OH
OH
OH
HO
O
HO
O
HO
HO
O
O
HO
O
MeO
MeO
O
O
F3’H
F6H
F6MT
F7MT
MeO
OH
OH
OH
OH
OH
OH
O
OH
OH
O
OH
O
OH
Quercetin
Quercetagetin
Patuletin
F7GT
Eupatoletin
Kaempferol
F7GT F3GT
F7GT F3GT
F7GT
OH
OH
OH
OH
OH
OH
OH
OH
OH
MeO
MeO
O
R
1
O
O
GluO
HO
O
GluO
MeO
O
R
1
O
O
F6H
F6MT
ORha
OR
OH
OH
OR
OH
O
OH
O
OH
O
OH
O
OH
O
R1: Glu, R=H: Km7glu
R1=H, R=Glu: Km3glu
R1: Glu, R=H: Qu7glu
R1=H, R=Glu: Qu3glu
Quercetagetin 7glu
Patulitrin
Eupatolin
Fig. 7. Biochemical pathway leading to the flavonols in R. hirta.
3
.3. Chemicals
the laboratory collection of M. Luczkiewicz. 6-OHKm, 6-MeOKm
and Quag3glu came from E. Wollenweber (TU Darmstadt, Ger-
many). The cytochrome P450 reductase-specific antiserum from
Jerusalem artichoke was a gift from F. Durst and I. Benveniste
(CNRS, Strasbourg, France). NADPH, cytochrome c, glucose oxidase
and catalase were obtained from Sigma (Vienna, Austria). Cyto-
chrome P450-specific inhibitors were a gift from W. Rademacher
(BASF, Limburgerhof, Germany).
UDP-D-[U-14C]glucose (12.0 GBq/mmol), S-adenosyl-L-
1
4
14
[
methyl- C]methionine (2.15 GBq/mmol) and [2- C]malonyl-
coenzyme A (2 GBq/mmol) were purchased from GE Healthcare
(
(
Freiburg, Germany). Non-labelled UDPG, SAM and malonyl-CoA
all Sigma-Aldrich, Vienna, Austria), respectively, were added to
1
4
their corresponding [ C]compounds to achieve the specific activi-
14
ties indicated in the enzyme assay section. [ C]Flavonoids were
prepared as previously described (Halbwirth et al., 2004, 2006).
Km, Km3glu, Km7glu, Qu, Qu3glu, Quag and Quag7glu, were pur-
chased from Extrasynthese (Genay, France), 7-MeQu and 3-MeQu
from Fluka (Vienna, Austria) and Qu7glu, 3,7-DiMeQu, 6-MeOQu,
and 6-MeOQu 7glu from Apin Chemicals (Abingdon, UK). 6,7-Di-
MeOQu, 6,7-DiMeOQu3rha, and 6,7-DiMeOKm3rha were from
3.4. Reflectance spectroscopy
The reflectance curves of the flower petals were obtained in the
spectral region between 300 and 700 nm as the fraction of the light
spectrum reflected by the sample in relation to the light spectrum
reflected by an ideally white, diffuse reflecting surface. The illumi-
Table 1
(Addition to Fig. 4). Maximal UV reflectance (%) of apical and basal petal parts and the corresponding wavelength positions for the front side and the rear side in four different
anthesis stages.
Anthesis stage
1
2
3
4
Frontal side
Apex
Max. UV-reflectance (%)
wavelength number
Max. UV-reflectance (%)
wavelength number
14.3 ± 1.0
367.3
13.5 ± 2.6
367.3
13.0 ± 1.0
367.3
11.8 ± 1.8
367.3
15.5 ± 2.1
356.6
10.0 ± 1.1
356.6
16.2 ± 1.8
356.6
8.5 ± 0.7
356.6
Base
Rear side
Apex
Max. UV-reflectance (%)
Wavelength number
Max. UV-reflectance (%)
Wavelength number
24.3 ± 2.2
367.3
25.7 ± 4.0
367.3
19.1 ± 2.3
367.3
21.1 ± 5.5
367.3
21.5 ± 2.0
374.4
20.3 ± 5.3
374.4
19.5 ± 1.5
372.0
24.0 ± 3.4
372.0
Base

K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
897
Table 2
Dependence of the F6H on cofactors and influence of potential enzyme inhibitors.
3.7. Enzyme preparations and protein determination
Additions
Relative activity (%)
Crude enzyme preparations and microsomal preparations from
petals of R. hirta were obtained as described earlier (Halbwirth
et al., 2006), using buffer 2. To remove low molecular compounds,
crude enzyme preparations were passed through a gel chromatog-
raphy column (Sephadex G25, GE Healthcare, Freiburg, Austria).
Protein content was determined by a modified Lowry procedure
None
NADPH, 1.45 mM
NADH, 1.45 mM
NADPH,1.45 mM + FAD, 1
NADH, 1.45 mM + FAD, 1
Oxygen consuming system
Oxygen consuming system, heat inactivated
Ketoconazol, 50 mM
Tetcyclacis, 50 mM
Ancymidol, 50 mM
EDTA, 5 mM
0
100
20
113
28
15
104
18
11
70
100
0
lM
lM
(
Sandermann and Strominger, 1972) using crystalline bovine ser-
um albumin as a standard.
3.8. Enzyme characterization
Cyt. P450 red. specific antibodies, 10
Cyt. P450 red. specific antibodies, 10
l
l
g
g inactivated
98
All data represent an average of at least three independent
experiments. Subcellular determination was carried out according
to Halbwirth et al. (2004). Determination of the pH optimum was
carried out as described for the standard assay, but using 0.2 M
buffers with pH values between 6.0 and 8.5. Exclusion of oxygen
from the enzyme assay was carried out using an oxygen consum-
ing system consisting of glucose, glucose oxidase and catalase
according to Wimmer et al. (1998).
All activities were expressed per kg of total protein and relative activities were
calculated compared to the activity of the microsomal fraction obtained in the
presence of 1.45 mM NADPH. 100% correspond to 0.2 lkat/kg protein.
nation of the samples was accomplished using a 75 W ozone-free
Xenon arc lamp (Oriel 6251) as white light source (color tempera-
ture 5500 K). An F/1.0 quartz condenser (Oriel 68806) was used to
obtain a collimated light beam, which passed an infrared blocking
filter (Oriel 6123) for avoidance of thermal stress. The petal sam-
ples were fixed on an opaque black holder (non-reflecting back-
ground) with the help of an opaque black mask having a
3.9. Enzyme assays
In a final volume of 100
enzyme preparation (29–151
CoA (1.5 nmol, 1300 Bq), 5
50 l buffer 1; the FHT assay contained 0.046 nmol [ C]naringenin
(108 Bq), 30 l enzyme preparation (9–56 g total protein), 5
3.48 mM 2-oxoglutarate (aqueous), 5
l 2.01 mM FeSO ꢁ 7H O
(aqueous), and 80 l buffer 2; the FLS assay contained 0.046 nmol
C]DHK (108 Bq), 40 l enzyme preparation (29-151 g total pro-
tein), 5 l 3.48 mM 2-oxoglutarate (aqueous), 5
O ꢁ 7H O (aqueous), and 50
ll: the CHS/CHI assay contained 40 ll
14
lg total protein), 5
ll [ C]malonyl-
3
ꢁ 3 mm hole, the latter allowing the illumination of a selected
ll p-coumaroyl-CoA (1.0 nmol), and
1
4
area of the petal. The light diffusely reflected by the petal was fo-
cused through a quartz lens (f = 140 mm) into the entrance slit of
a grating spectrometer (Jobin Yvon Triax 190), spectrally dispersed
by a diffraction grating with 300 lines mm , and detected by a
Peltier cooled CCD camera (Jobin Yvon) at the exit aperture of
the spectrometer. As reference sample a white, diffuse reflecting
standard (Spectralon TM SrS-99-010, 99% reflectance from 300 to
l
l
l
ll
l
4
2
ꢀ1
l
1
4
[
l
l
l
2
l
l 2.01 mM FeS-
0
4
ll
buffer 2; the F3 H assay
1
4
1
200 nm) was used. Determining the ratio of the sample spectrum
contained 0.046 nmol [ C]naringenin (108 Bq), 20
preparation (6.5–44 g total protein), 5 l 1.30 mM NADPH (aque-
ous), and 75 l buffer 1; and the F6H assay contained 0.046 nmol
C]Qu (108 Bq), 40 l microsomal preparation (27–112 g total
protein), 5 l 1.45 mM NADPH (aqueous) and 55 l buffer 3. In a
final volume of 50 l: the MT assay contained 20 l enzyme prep-
aration (29–151 g protein), 30 nmol flavonoid substrate (dis-
solved in EGME), 1.45 mM MgCl2 (aqueous),
C]SAM (0,43 nmol, 925 Bq, aqueous), and 20 l buffer 2 and
the GT assay contained 20 l enzyme preparation (15–75 g total
l [ C]UDPG (0.2 nmol, 1500 Bq), 15 nmol flavonoid
substrate (dissolved in 2.5 l ethylene glycol mono methyl ether),
and 25 l buffer 1.
The assays were incubated for 30 min at 30 °C. PAL, CHS/CHI
and MT assays were stopped with 200 l ethyl acetate and
10 l acetic acid and the amounts of product formed were deter-
mined on a scintillation counter. For MT assays, the remaining or-
ganic phase was applied to cellulose plate and
ll enzyme
divided by the reference automatically eliminates the necessity for
grating efficiency correction and spectral sensitivity correction of
the CCD.
l
l
l
1
4
[
l
l
l
l
3.5. Chromatography
l
l
l
Substrates and products were separated by TLC on Merck pre-
coated cellulose (without fluorescence indicator, 1.0571.001,
VWR International, Vienna, Austria) using the following solvent:
5
ll
5
ll
5 ll
1
4
[
l
l
l
1
4
(
1) n-butanol/acetic acid/water (6:1:2, v/v/v); (2) chloroform/ace-
protein), 2.5 l
tic acid/water (10:9:1, v/v/v) (3) 30% (v/v) acetic acid. Product
identification was performed by HPLC analysis according to Kim
et al. (2005) using a Perkin Elmer Series 200 system equipped with
a photo diode array detector coupled with a 500TR Flow Scintilla-
l
l
l
tion Analyzer for the detection of radiolabelled substances (R
MeO Qu7glu: 4.40; R Qu3glu: 4.80; R Qu7glu: 5.30; R Quag7glu:
7-MeQu: 8.53; R 7-MeQu: 9.40; R Quag: 9.74; R 6-MeQu:
1.08; R 6,7-DiMeOQu: 12.5; R Qu: 13.58). 6-MeO Qu7glu, Quag7-
t
6-
l
t
t
t
6
.90; R
t
t
t
t
a
1
t
t
chromatographed with solvent system 2 to separate the different
0
glu, Qu7glu and Qu3glu were purified via paper chromatography in
solvent 3 before they were subjected to HPLC analysis. LC-UV-ESI-
MSn analysis was performed according to Lunkenbein et al. (2006).
products formed. FHT, FLS, F3 H and F6H assays were terminated
by the addition of 70
ll ethyl acetate and 10 ll acetic acid. To FLS
assays 10 l of 0.1 mM EDTA was also added before the extrac-
l
tion. The organic phases were transferred to a precoated cellulose
3.6. Buffers used
plate (Merck, Germany). After developing the TLC plates in sol-
vent system 2 (FHT, FLS, and F3 H) or solvent 1 (F6H) conversion
0
The following buffers were used for the enzyme assays: buffer 1
rates were determined with a TLC linear analyzer. GT assays were
terminated by the addition of 10 ll acetic acid and 25 ll metha-
0
(
CHS, F3 H and GT assays): 0.1 M KH
2
PO
/K
4 2
HPO
4
(containing 0.4%
Na-ascorbate), pH 7.5; buffer 2: (FHT, FLS and MT assays): 0.1 M
Tris/HCl (containing 0.4% Na-ascorbate), pH 7.6; buffer 3 (F6H as-
nol. The mixture was chromatographed on Schleicher and Schüll
2043b paper in solvent 3. The zones containing the labelled prod-
ucts were cut out and radioactivity was quantified on a scintilla-
tion counter.
2 4 2 4
says): 0.1 M KH PO /K HPO (containing 0.4% Na-ascorbate), pH
7
.0.

8
98
K. Schlangen et al. / Phytochemistry 70 (2009) 889–898
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Acknowledgements
Chemistry and Biochemistry of Plant Pigments, vol. 1. Academic Press, London,
pp. 736–778.
These investigations were supported by a grant from the Aus-
trian FWF (Project P-17629-B03). H. Halbwirth acknowledges her
grant from the Austrian FWF (Project V18-B03). We are indebted
to E. Wollenweber (TU Darmstadt, Germany) for providing us with
a multitude of reference compounds. We are grateful to F. Durst
and I. Benveniste (CNRS, Strasbourg, France) for their gift of cyto-
chrome P450 reductase-specific antiserum, to Jenny Greenham
Harborne, J.B., 1988. The flavonoids: recent advances. In: Goodwin, T.H. (Ed.), Plant
Pigments. Academic Press, London, pp. 299–344.
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(
University of Reading, UK) for kindly providing us with reference
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