- Email: [email protected]
Identification, Properties and Genetic Control of UDP-Glucose : Cyanidin 3-0-Glucosyltransferase in Petunia hybrida
Department of Plant Physiology, University of Amsterdam, Amsterdam, The Netherlands, and Department of Population and Evolutionary Biology, University of Utrecht, Utrecht, The Netherlands
Identification, Properties and Genetic Control of UDP-Glucose : Cyanidin 3-0-Glucosyltransferase in Petunia hybrida K. F. F.
KHO,
J. KAMSTEEG and J. VAN BREDERODE
With 15 figures Received 27 February 1978 . Accepted 11 March 1978
Summary In corollas of Petunia hybrida the presence of a glucosyltransferase capable of catalyzing the transfer of glucose from UDP-glucose to the 3-0-position of cyanidin was demonstrated. The enzyme could also use delphinidin as a substrate. The glucosyltransferase exhibited a pH optimum between 7.5-8.0 and had a «true Km» value of 1.7 mM for UDP-glucose. For the substrates cyanidin and delphinidin allosteric behaviour was observed. The Hili number was determined to be 1.9 for cyanidin. The K' values were 2.5 X 10-3 mM for cyanidin and 5 X 10-3 mM for delphinidin. Cyanidin 3-glucosylation was inhibited by delphinidin and delphinidin 3-glucosylation was inhibited by cyanidin, either inhibition being of the uncompetitive type. The enzymatic activity was controlled by the genes An1 and An2. Genotypes in which either of these genes was in the homozygous recessive state showed 20 0/0 of the activity of the control value, without changes in the substrate affinities of the enzyme.
Key words: glucosyltransjerase, cyanidin, genetic control, Petunia hybrida.
Introduction Genetic studies of flower colour in Petunia hybrida demonstrated that several genes are involved in the regulation of flavonoid biosynthesis (WIERING, 1974). The synthesis of anthocyanins is governed by the complementary genes An1, An2, and An3. Dominant alleles of each of these genes are required for normal synthesis of cyanidin 3-glucoside in the flower limb. It has been demonstrated that white flowers of the genotype AnlAnlAn2An2an3an3 are capable to convert exogeneous supplied dihydroquercetin into cyanidin 3-glucoside. The genotypes anlanlAn2An2An3An3 and AnlAn1an2an2An3An3 are unable to do this. Moreover, the latter genotypes accumulate dihydroquercetin and related dihydroflavonols in the flower (Figure 1, cf. also KHO et al., 1975, 1977). Obviously, dominant alleles of Anl and An2 are required for the conversion of
z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
450
K.
F. F.
KHO,
J.
KAMSTEEG
J.
and
VAN BREDERODE
r----------, 1
An1, An 2
I OH
OH
1
0
1HO
OH
1
An3
OH
0
--I,\~
I
1 1
1 HO
1
(\1
OH
1 OW
UDPG
~
UDPI
L ________ ....l dihydroflavonoL intermediates
cyanidin
cyanidin - 3 -gLucoside
Fig. 1: Genetic control of cyanidin 3-glucoside biosynthesis in Petunia hybrida. Dominant alleles of An3 are required for synthesis of dihydroflavonol intermediates. The conversion of dihydroquercetin (R = H) into cyanidin 3-glucoside is governed by the genes Anl and An2. This conversion requires at least two re action steps, probably via cyanidin as intermediate. The glucosylation at position 3 may be catalyzed by a glucosyltransferase.
dihydroquercetin into cyanidin 3-glucoside. Dihydroquercetin is a weIl known precursor of anthocyanins and flavonols (PATSCHKE and GRISEBACH, 1968; FRITSCH, and GRISEBACH, 1975; STICKLAND and HARRISON, 1974). The conversion into cyanidin 3-glucoside requires at least two reaction steps: (i) conversion of the dihydroflavonol into the corresponding anthocyanidin by intramolecular rearrangement and removal of a hydroxyl ion (JURD, 1969) and (ii) a glucosylation at position 3. The correct sequence of these reactions has not yet been established. But there is some evidence that the glucosylation re action is the last step. (c.f. Fig. 1, FRITSCH and GRISEBACH, 1975; RIBEREAU-GAYON, 1972). The present work describes the properties ofUDP-glucose: cyanidin 3-0-glucosyltransferase. The presence of this enzyme has been reported in Silene dioica by VAN BREDERODE (1975) and in red cabbage and Haplopappus gracilis bei SALEH et al. (1976 a, 1976 b). The enzyme is responsible for the 3-glucosylation reaction outlined in Fig. 1. and we undertook experiments to deterrriine whether the enzyme is controlled by the genes An1 or An2. Materials and Methods Plant Material The red flowering Petunia line R3 (genotype AnlAnlAn2An2An3An3) and the white flowering mutants W39 (genotype AnlAnlAn2An2an3an3), W38 and W19 (both with genotype anlanlAn2An2An3An3) and W12 and A10 (both with the genotype AnlAnlan2an2An3An3) were cultivated in the greenhouse of the Institute of Genetics, University of Amsterdam. Chemieals UDP-(U-14C)-glucose, 233 Ci/mole and ADP-(U 14C)-glucose, 283 Ci/mole were supplied by the Radiochemical Centre, Amersham, England. UDP-glucose and ADP-glucose were Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
451
purchased from Sigma. The specific activity of the nucleotide sugars was adjusted by addition of carrier to ca. 1 Ci/mole. For determination of specific activity and chemical purity see VAN BREDERODE and VAN NIGTEVECHT (1973'). Cyanidin chloride was synthesized according to KRISHNAMURTY et al. (1962) and purified by preparative chromatography on Whatman-3 paper in the solvent systems butanol-1 acetic acid - water (4: 1 : 5, viviv, upper phase) and 10f0 w/v HCI. Finally, the anthocyanidin was precipitated as an amorphous solid with diethylether. Delphinidin chloride was obtained from Carl Roth A.G., Karlsruhe, Federal Republic of Germany. The 3-glucosides of cyanidin and delphinidin were isolated from appropriate genotypes of Petunia hybrida (WIE RING and DE VLAMING, 1973). Pro tein Assay
Protein was determined according to the method of LOWRY et al. (1951) using bovine albumin as a standard. Enzyme Preparation
All steps were performed between 0 and 4°C. From flower buds of stage III (KHO et al., 1975) the limb part was dissected. One gram of the flower limb material was homogenized in an all glas Pott er homogenizer in 5 ml 50 mM sodium-potassium-phosphate buffer pH 7.5, containing 5 0/0 soluble polyvinylpyrrolidone and 20 mM ß-mercaptoethanol. The homogenate was centrifuged for 10 min at 38,000 X g. To remove endogenous substrate, phenolic substances, soluble PVP and low molecular weight materials, the supernatant was passed through a polyclar AT column (20 X 1 cm) (KAMSTEEG and VAN BREDERODE, 1978) and subsequently through a Sephadex G-50 column (30 X 2.5 cm). The polyclar AT and Sephadex G-50 columns were equilibrated and eluated with 10 mM sodium-potassium phosphate buffer pH 7.5, containing 4 mM ß-mercaptoethanol. Unless noted otherwise the high molecular weight fraction of the Sephadex G-50 eluate was used as an enzyme source. Enzyme Assays
The standard re action mixture consisted of 50 ,ul enzyme, 5 ,ul 10 mM cyanidin, dissolved in 5 mM HCl and 15,u1 10 mM UDP-glucose, labelIed uniformly in the glucose moiety (S.A. ca. 1 Ci/mole). The reaction mixture was incubated for 5 min at 30°C, unless specified otherwise. The re action was stopped by addition of an equal volume of 1 % (w/v) trichloroacetic acid (TCA) in methanol. After addition of carrier cyanidin 3-glucoside, the re action mixture was transferred quantitatively as a spot on Whatman-3 paper and developed two dimensionally in the solvent systems butanol-1-acetic acid water (4 : 1 : 5, v/v/v, upper phase) and 1 % (w/v) HCI respectively. After drying, the spot corresponding to cyanidin 3-glucoside was cut out and placed in a scintillation vial. After addition of scintillation solvent, composed of 4 g 2,5-diphenyloxazole and 50 mg 1,4-bis-2-(5-phenyloxazoyl)-benzene in 1 I toluene, the vial was counted in a Packard liquid scintillation spectrometer (counting yield approximately 80 %). For the determination of zero time control, TCA was added to the reaction mixture before incubation. UDP-glucose : delphinidin 3-0-glucosyltransferase activity was determined by the same procedure as described for cyanidin, using delphinidin as substrate and delphinidin 3-glucoside as a carrier. In some cases cyanidin and delphinidin were tested simultaneously. In this case incorporation of radioactivity in cyanidin 3-glucoside and delphinidin 3-glucoside was measured by addition of both carriers to the reaction mixture and determination of radioactivity in the spots of the 3-glucosides after two dimensional chromatography. Radioactivity found in case of zero time controls was between 0-10 dpm above the background (25-30 dpm) found with an untreated piece of Whatman-3 paper. All enzyme tests were run in duplicate or triplicate. Z. Pjlanzenphysiol. Bä. 88. S. 449-464. 1978.
452
K. F. F.
KHO,
J.
KAMSTEEG
and
J.
VAN BREDERODE
Results
1. Enzymatic Synthesis
0/ cyanidin 3-glucoside
Incubation of UDP-glucose, labelled uniformly in the glucose moiety, with cyanidin and an enzyme preparation from flower buds of the red flowering line R3 - in which the An genes are dominant - resulted in incorporation of radioactivity in cyanidin 3-glucoside. When the enzyme was omitted in the test, or TCA was added before incubation, no incorporation was observed (Table 1). Table 1: Synthesis of cyanidin 3-glucoside from cyanidin and UDP-glucose, catalyzed by an enzyme preparation from flower buds of Petunia hybrida. enzyme fraction
substrate added
additives
dpm
38,000 Xg supernatant
cyanidin none cyanidin cyanidin
none none 0.1 % tween 80 15mM EDTA
3308 1431 2918 1512
polyc1ar AT + sephadex G 50 elutae
cyanidin none cyanidin cyanidin
none none 6 mM MgCl 2 6mM MnCl 2
1568 32 1215 612
none
cyanidin
phosphate buffer pH 7.5
23
polyc1ar AT + Sephadex G 50 eluate
cyanidin
50 ,ul 1 Ofo TCA in methanol
26
Activity was expressed as dpm incorporated in cyanidin 3-glucoside. The nuc1eotide sugar was labelIed with a specific activity of ca. 1 Ci/mole. Enzyme preparations were obtained from the red flowering line R3. The enzyme tests were carried out as described in Materials and Methods.
When cyanidin was omitted in the test of the supernatant of the crude homogenate, about half of the product formation remained, indicating that the supernatant contained endogenous cyanidin. When the supernatant was passed through polyclar AT and Sephadex G-50 columns subsequently, only the high molecular weight fraction proved to be active in the enzyme test. Omitting of cyanidin in this case gave no activity, indicating that the endogenous cyanidin was completely removed. Addition of detergent (Tween 80, 0.1 Ofo final concentration) did not enhance the enzymatic activity. The product formation was reduced by EDTA, but could not be stimulated by divalent metal ions, such as Mg 2+ and Mn 2+ (Table 1). The partial purification of the enzyme is further outlined in Table 2. This Table shows that considerable activity was left in the pellet, which could not be washed out with buffer.
z. Pflanzenphysiol.
Bd. 88. S. 449-464. 1978.
453
UDP-glucose : cyanidin 3-0-glucosyltransferase
Table 2: Purification of UDP-glucose: cyanidin 3-0-glucosyltransferase isolated from the corolla of Petunia hybrida. fraction
protein (mg)
units
crude homogenate
48
119
2.5
54
38,000 Xg pellet rinsed with buHer
22
79
3.6
36
38,000 Xg supernatant
16
219
14
100
92
42
42
Polyclar AT + Sephadex G-50 eluate
2.2
specific activity
recovery
enzyme purification
0/0
5.6 17
One enzyme unit is defined as the amount of enzyme which catalyzes the formation of 1 nmole product per minute at 30 oe in the enzyme assay.
The partial purification of the enzyme is further outlined in Table 2. The specificity of the enzyme test for cyanidin 3-glucoside formation was checked previously by KAMSTEEG and VAN BREDERODE (1978). The quantity of cyanidin 3-glucoside formed from cyanidin and UDP-glucose was proportional with time up to 10 min (Figure 2). The leveling off of the curve after 10 min is probably caused by inactivation of the cyanidin at the pH used. Preincubation of the enzyme for 30 min with UDP-glucose and stopping the re action 5 min after the addition of cyanidin resulted in an equal amount of product forn;'ation as when both substrates were added simultaneously. N
I~
15
x
E
Q.
Fig. 2: Formation of cyanidin 3-glucoside from cyanidin and UDP-glucose as a function of time. The incubations were carried out under standard conditions as described in Materials and Methods.
o
2 5
10
20
30 minutes
Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
454
K.
F. F.
KHO, }. KAMSTEEG
and J.
The product formation was found enzyme (Figure 3).
to
VAN BREDERODE
be proportional to the amount of added
E 1200
0.
"
800
400
o
10
20
30
40
50
Jll
enzyme
Fig. 3: Formation of cyanidin 3-glucoside from cyanidin and UDP-glucose as a function of added enzyme. The enzyme was assayed as described in Materials and Methods. The pH optimum was determined to be in the range 7.5-8.0 (Figure 4). In connection with the pH range of the phosphate buffer further tests were run at pH 7.5. N
'~ x
15
E
J
a.
"t:>
10
5
5
6
7
B
9
pH
Fig. 4: Effect of pH on UDP-glucose: cyanidin 3-0-glucosyltransferase. The reaction mixture contained in a total volume of 107 .u1: 50 .ug protein, 12.5 ,umoies potassiumsodium phosphate, 200 nmoles ß-mercaptoethanol, 240 nmoles UDP-(U14C)-glucose (S. A., 1 Ci/mole), 83 nmoles cyanidin. Incubation mixtures prepared in parallel (cyanidin and UDP-glucose omitted) were used to determine pH.
The results indicate clearly the presence of an enzyme, capable of catalyzing the transfer of the glucose moiety from UDP-glucose to the 3-0-position of cyanidin in flower limbs of Petunia hybrida.
2. Substrate Specificity The enzyme was also capable to catalyze the transfer of the glucose moiety from UDP-glucose to the 3-0-position of delphinidin (Table 3). Neither for cyanidin, nor for delphinidin ADP-glucose could serve as a glucose donor. For delphinidin glucosylation a similar pH optimum as mentioned for cyanidin (7.5-8.0) was observed.
z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
455
Table 3: Substrate specificity of UDP-glucose : cyanidin 3-0-glucosyltransferase of Petunia hybrida. Sugar donor
acceptor
carrier
radioactivity cochromatographed with the carrier (dpm)
UDP-glucose
cyanidin
2885
UDP-glucose
delphinidin
ADP-glucose
cyanidin
ADP-glucose
delphinidin
cyanidin 3-glucoside delphinidin 3-glucoside cyanidin 3-glucoside delphinidin 3-g1ucoside
2619
3 5
The enzyme was isolated from genotype R3 and purified by filtration over polyclar AT and Sephadex G-50 columns. The re action mixture contained 500 nmoles potassium-sodium phosphate buHer, pH 7.5, 200 nmoles ß-mercaptoethanol, 50 nmoles of UDP-(U 14C)glucose (S.A. 6 Ci/mole) or ADP-(U-14C)-glucose (S.A. 6 Ci/mole), 50 nmoles of the anthocyanidin and 16 flg protein. Radioactivity was determined in the spot of the carrier substance after cochromatography with the re action mixture as described in Materials and Methods and given as dpm above the background (25-30 dpm).
3. Glucosyltransferase Activity in Various Stages of Flower Bud Development In the vegetative parts of the plant no enzymatic activity could be detected. Figure 5 shows that the glucosyltransferase activity started to develop after initiation of the flower bud and could be detected in the corolla. The highest activity was present in bud stage III, according to KHO et al. (1975). This stage is correlated with rapid ..
~
150
...
Cl
I
.~ 100
Fig. 5: Activity of the glucosyltransferase in various stages of the developing flower budo For activity measurements 38,000 X g supernatants were used. Assays were carried out as described in Materials and Methods. Bud length is defined as the distance between the flower receptacle and the top of the corolla (KHO et al., 1975). Stage I: 0-5 Olm, Stage 11: 5-15 Olm, Stage 111: 15-35 Olm, Stage IV, 35 mmopen flower, Stage V: open flower, G: vegetative material.
vi
GI
"0 E I:
50
o...L..=._'::l-n-':='---':::I-...I....L.--Ln~ G I :n ][ :nz: J[ bud stage
z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
456
K.
F. F.
KHO,
J.
KAMSTEEG
and }.
VAN BREDERODE
elongation of the tubular corolla and with a high rate of anthocyanin synthesis. During later stages of flower life the glucosyltransferase activity decreased markedly. 4. Kinetics LINEWEAVER and BURK (1934) plots of the reciprocal of the initial velocity of the cyanidin glucosylation reaction versus the reciprocal of the UDP-glucose concentration at different fixed cyanidin concentrations show that the relationship between V-i and [UDP-glucose]-i is virtually linear, the apparent Km value being dependent upon the second substrate (cyanidin) concentration (Figure 6).
v- 1
30
1.0
2.0
3.0
[UDPGr 1 mM- 1
Fig. 6: Lineweaver & Burk plot of the reciprocal of the initial velocity of the cyanidin 3glucosylation re action versus reciprocal of mMolar concentration of UDP-glucose. The following concentrations of cyanidin were used: 0.0308 mM (0--0); 0.0431 mM (e-e); 0.0631 mM (0-0); 0.1369 mM ( . - . ) . The reaction mixture contained in a total volume of 65,u1: 500 nmoles potassium-sodium phosphate buffer pH 7.5, 200 nmoles ßmercaptoethanol, 8,ug protein, the indicated amount of cyanidin and UDP-glucose in a concentration ranging from 0.36 to 0.89 mM.
When the reciprocal of the initial velocity was plotted versus the reciprocal of the cyanidin concentration at different fixed UDP-glucose concentrations, no linear relationship between V-i and [cyanidin]-i was observed. However, when the reciprocal of the initial velocity was plotted versus the reciprocal of the square of the cyanidin concentration, a virtually linear relationship was obtained (Figure 7). These kinetic responses indicate that the enzyme behaves as a Michaelis-Menten system with respect to UDP-glucose, but as an allosteric system (SEGEL, 1974 a) with respect to the cyanidin substrate. A secondary plot according to FLORINI and VESTLING (1951) of the intercepts on the V-i axis of Figure 6 versus the reciprocals of the square of the fixed cyanidin concentrations yielded the Vmax and the K' value for cyanidin. A secondary plot of the intercepts on the V-i axis obtained from Figure 7 versus the reciprocals of the fixed UDP-glucose concentration yielded the «true Km» value for
z.
Pflanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
~ I
457
20
E
Co
"C
400
800
1200
[CVANIDINr 2
mM- 2
Fig. 7: Plot of the reciprocal of initial velocity of the cyanidin 3-glucosylation reaction versus reciprocal of the square of mM concentration of cyanidin. The following concentrations of UDP-glucose were used: 0.36 mM (0-0); 0.45 mM (e-e); 0.54 mM (0--0); 0.89 mM ( . - - . ) . The reaction mixture contained in a total volume of 65.ul: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles p-mercaptoethanol, 8.ug protein, the indicated amount of UDP-glucose and cyanidin in a concentration ranging from 0.0308 to 0.1369 mM.
-1 V max 10 I
E
Co
"C
Km -1 1.0 I
800 (K,)-1
mM-1
[UDPG]-1 0 I
400
I
0
2.0
1.0 I
400
I
800
[CVANIDINr 2
3.0 I
1200 mM- 2
Fig. 8: Plot of the intercepts on the vertical axis of the data plotted in Figures 6 and 7 (corresponding to lIvmax in the presence of the second substrate as the limiting factor), replotted versus reciprocal of corresponding fixed UDP-glucose concentration (0-0) or reciprocal of the square of fixed cyanidin concentration (e-e). The intercept on the vertical axis equals l/vmax and the intercepts on the horizontal axis equal l/true Km for UDP-glucose an.d l/K' for cyanidin. Z. Pjlanzenphysiol. Bd. 88. S, 449-464. 1978.
458
K. F. F. KHO,
J. KAMSTEEG and J. VAN BREDERODE
UDP-glucose. Both secondary plots are given in Figure 8. The kinetic parameters are listed in Table 4. The kinetics of delphinidin 3-0-glucosylation was determined in a similar manner as mentioned for cyanidin 3-0-glucosylation and is presented in the Figures 9, 10 and 11. The relationship between V-i and [delphinidinJ-2 was also virtually linear. The kinetic parameters of this reaction are also listed in Table 4. The kinetic responses for cyanidin indicate a Hill number (SEGEL, 1974 a) of the enzyme elose to 2.0 for anthocyanidin substrates and elose to 1.0 for UDP-glucose. Direct determination of the HilI number by HilI plots (BOURDILLON et al., 1976) Table 4: Kinetic parameters for enzymatic transfer of glucose from UDP-glucose to the 3-0 position of cyanidin or delphinidin by a purified enzyme preparation from flower buds of the red flowering line R3 of Petunia hybrida. The data were obtained from the plots given in the figures 8 and 11. K'b) delphinidin (mM)
reaction
V max (nmoies . min- 1 . mg protein-1 )
Kma) UDP-glucose (mM)
K'b) cyanidin (mM)
cyanidin 3-glucosylation delphinidin 3-glucosylation
69
1.70
2.5 X 10-3
54
1.02
5.0 X 10-3
a) «True» Michaelis constant. b) K' is defined as the substrate concentration at ~ Vmax raised Hill number (cf. SEGEL, 1974 a).
to
apower equal to the
v- 1 10
1.0
2.0 [UPDGr 1
3.0 mM- 1
Fig. 9: Lineweaver & Burk plot of the initial velocity of the delphinidin 3-glucosylation re action versus reciprocal of mM concentration of UDP-glucose at fixed concentrations of delphinidin. The following delphinidin concentrations were used: 0.0383 mM (e-e); 0.0585 mM (0-0); 0.0802 mM ( . - . ) ; 0.1185 mM (0-0). The reaction mixture contained in a total volume of 65,u1: 500 nmoles potassium-sodium phosphate buffer, pH 7.5, 200 nmoles ß-mercaptoethanol, 13.5,ug protein, the indicated amount of delphinidin and UDP-glucose in a concentration ranging from 0.374-0.892 mM. Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
200
400
600
[DELPHINIDINr 2
459
BOO
mM- 2
Fig. 10: Plot of the recipocal of initial re action velocity of delphinidin 3-glucosylation versus reciprocal of the square of mM concentration of delphinidin at fixed concentrations of UDP-glucose. The following UDP-glucose concentrations were used: 0.374 mM (e---e); 0.552 mM (0-0); 0.892 mM (0-0). The reaction mixture contained in a total volume of 651ll: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles ß-mercaptoethanol, 13.5 flg protein, the indicated amount of UDP-glucose and delphinidin in a concentration ranging from 0.0383'---0.1185 mM. V -1 max 10
'I E Co
...."tJ ~
5
Km -1
[UDPG]-1 mM- 1 1.0
0 I
300 200
(KT 1
I
100
I
0
2.0
1.0 I
100 200
I
300
I
I
3.0 I
400 500 600
I
700
[DELPHINIDI N] - 2 mM- 2
Fig. 11: Plot of the intercepts on the vertical axis of the data plotted in Figures 9 and 10 (corresponding to l/vmax in the presence of the second substrate as the limiting factor) replotted versus reciprocal of corresponding fixed UDP-glucose concentration (0---0) or reciprocal of the square of corresponding fixed delphinidin concentration (e---e). The intercept on the vertical axis equals l/vmax and the intercepts on the horizontal axis equal lItrue Km for UDP-glucose and lIK' for delphinidin.
proved to be unsatisfactory (TABAK, personal communication), because V max cannot be determined directly, as the enzyme tends to be inhibited to some extent at high anthocyanidin concentrations. Therefore we determined the Hill number by another method. We calculated the correlation coefficient for linear regression between V-i and [substrate]-n in case of cyanidin and UDP-glucose. The value of n that gives the highest correlation coefficient is equivalent to the Hill number (SEGEL, Z. Pflanzenphysiol. Bd. 88. S. 449-464. 1978.
460
c.
K.
F. F.
KHO,
J.
KAMSTEEG
and
J.
VAN BREDERODE
lOOO
'(3
:::.. o
u
.:5
:;;
0.990
~o u
0.980
(e_)
0.970
o
Fig. 12: Correlation coefficients for linear regression between v-1 and [substrate concentration]-n as a function of n for cyanidin and for UDP-glucose (0--0). The value of n corresponding to the highest correlation coefficient equals the Hill number for the substrate.
lO
1.5 hill
2.0
2.5
number
1974 a). As can be seen in Figure 12, the value of the Hill number was dose to 1.0 for UDP-glucose and dose to 2.0 (1.9) for cyanidin.
5. Competition Experiments involving Simultaneous Reaction of Cyanidin and Delphinidin with UDP-glucose Our test system allows investigation of the influence of delphinidin on the 3-0-glucosylation re action of cyanidin and vice versa. When both cyanidin and delphinidin were incubated with UDP-(UßC)-glucose in the presence of the enzyme preparation from genotype R3 and the reaction mixture was chromatographed two-dimensionally together with the carrier substances cyanidin 3-glucoside and delphinidin 3-glucoside, radioactivity was incorporated in the spots corresponding to cyanidin 3-glucoside and delphinidin 3-glucoside. Plots of the reciprocal of the initial velocity versus the reciprocal of the square of the cyanidin concentration at various fixed delphinidin concentrations are given in Figure 13. The corresponding plots for delphinidin at various fixed cyanidin concentrations are given in Figure 14. The graphs indicate that the 3-0-glucosylation of cyanidin is uncompetitively inhibited by delphinidin and vice versa (SEGEL, 1974 b). At high concentrations of both substrates, the sum of both reaction velocities was virtually constant. 6. Genetic Control
The activity of the glucosyltransferase in flower limb extracts of various genotypes of Petunia hybrida is presented in Figure 15. In the white flowering mutant W39 (genotype An1An1An2An2an3an3) the enzymatic activity proved to be in the same order as in the red flowering line R3 (genotype An1An1An2An2An3An3), which was used as a contro!. This indicates that the gene An3 is not involved in regulation of the glucosyltransferase activity, but controls probably another step in anthocyanin biosynthesis (cf. Figure 1). Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
461
v- 1 ..
E
...
20
Q.
500 [CVANIDIN]-2
1000 mM-2
Fig. 13: Plot of the reciprocal of initial reaction velocity of cyanidin 3-glucosylation versus reciprocal of the square of cyanidin concentration at fixed delphinidin concentrations: 0 mM 0.032 mM (0-0); 0.100 mM (0-0). The reaction mixture contained in a total volume of 75,ul: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles ji'-mercaptoethanol, 13.5,ug protein, 116 nmoles UDP-glucose, the indicated amount of delphinidin and cyanidin in a concentration ranging from 0.0397 to 0.0891 mM.
v- 1 ,. 10 E Q.
...
100
300
500
[DELPHINIDINr 2 mM-2
Fig. 14: Plot of the reciprocal of initial velocity of delphinidin 3-glucosylation versus reciprocal of the square of delphinidin concentration at fixed concentrations of cyanidin: o mM 0.025 mM (0--0); 0.077 mM (0-0). The re action mixture contained in a total volume of 75,ul: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles ji'-mercaptoethanol, 13.5,ug protein, 116 nmoles UDP-glucose, the indicated amount of cyanidin and delphinidin in a concentration ranging from 0.0492 to 0.1154 mM.
Genotypes with either An1 or An2 heing homozygous recessive showed a remarkahly lower enzymatic activity, not exceeding 20 Ofo of the line R3, used as a contro!. The mutants W19 and W38 are homozygous recessive for the gene An1 hut are different with regard to other genes, not related to the investigated part of the
z. Pjlanzenphysiol. Bd. 88. S. 449--464. 1978.
462
K. F. F.
KHO,
J.
KAMSTEEG
and
J.
VAN BREDERODE
'j'
-.
~ 20 CI
'j' c:
oe
ui
10
GI
-0
E c: R3
W 39
Fig. 15: Genetic control of glucosyl transferase activity in flower buds of Petunia hybrida. Activities were determined in 38,000 X g supernatants as described in Materials and Methods for cyanidin 3-0-glucosylation ( D ) and delphinidin-3-0-glucosylation (~ ). The following lines were investigated: R3 (genotype AnlAnlAn2An2An3An3); W39 (genotype AnlAn1An2An2an3an3); W38 (genotype an1anlAn2An2An3An3); W 19 (genotype anlanlAn2An2An3An3); W12 (genotype AnlAnlan2an2An3An3); AIO (genotype Anl Anl an2an2An3An3).
biosynthetic pathway. The genotypes W12 and AlO are homozygous recessive for the gene An2 and differ also in their genetic background (WIERING and DE VLAMING, unpublished results). The portein content in these genotypes was not seriously lowered and the specific activity of the enzyme did not exceed 30 % of the control value, estimated for R3. Kinetic studies revealed no significant differences of the substrate affinities for the enzymes present in genotypes homozygous recessive for either Anl or An2 or the control genotype R3, in which both genes are dominant. The results demonstrate a regulatory role of both genes Anl and An2 upon the total amount of the glucosyltransferase present in the flower limb of Petunia. Discussion Our results demonstrate clearly that the ultimate step of the biosynthetic route leading to cyanidin 3-glucoside has to be the glucosylation of cyanidin at position 3 in Petunia hybrida in agreement with the scheme postulated in Figure 1. An enzyme, capable of catalyzing this reaction, involving the transfer of a glucosyl residue from UDP-glucose to the 3-0-position of cyanidin has been detected in developing flower buds of Petunia hybrida. In vegetative plant material no such enzymatic activity could be detected. Th~ enzyme becomes detectable during the later stages of flower bud development, indicating that it is synthesized as a result of differential gene activity concerning flower development. The enzyme preparations were also capable to transfer glucose from UDP-glucose to delphinidin. However, in the genotypes investigated, cyanidin 3-glucoside is the major anthocyanin present. The partially purified enzyme preparations exhibited both for the substrates Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
463
cyanidin and delphinidin allosteric properties (SEGEL, 1974 a), and we determined the average HilI number to be 1.9. For the second substrate (UDP-glucose) we observed the common Michaelis-Menten type of kinetics (nh = 1.0). The kinetic constants (K') differ only slightly for cyanidin or delphinidin. As cyanidin is the normal substrate in our genotypes we designated the enzyme as a glucosyltransferase for cyanidin. The allosteric behaviour of the enzyme with regard to anthocyanidin substrates may be functional, since anthocyanidins are slightly soluble in aqueous environment and are probably produced on membranes (FRITSCH and GRISEBACH, 1975). The activation of the enzyme by a second binding site may be essential to achieve dissociation of the anthocyanidin from the membrane and simultaneous bin ding to the soluble enzyme. The soluble UDP-glucose however is randomly present in the environment of the enzyme and requires probably no special reaction mechanism for sufficient reaction rates. Thus, a Michaelis-Menten type reaction will ensure sufficient participation of UDP-glucose in the reaction. Competition experiments revealed that delphinidin inhibited the glucosylation of cyanidin and vice versa, but the sum of both activities was virtually constant. The inhibition resembles the uncompetitive model (SEGEL, 1974 b), suggesting that one enzyme is able to catalyze the 3-glucosylation of both anthocyanidins. Further evidence for this may be derived from the virtually constant ratio of both enzymatic activities in various genotypes and from the equal pH optima. The genes An1 and An2 are both involved in the regulation of the glucosyltransferase activity. When one of these genes is homozygous recessive, the enzyme activity is reduced to 20-30 Ofo of the original value, without significant differences of kinetic parameters. A genotype in which both genes are homozygous recessive could not be tested, because it was not available. The observed data indicate that both genes play a regulatory role with regard to the amount of glucosyltransferase activity present and are not to be considered as structural genes, controlling this activity. UDP-glucose: cyanidin-3-0-glucosyltransferases have been reported earlier by VAN BREDERODE et al. (1975) and by SALEH et al. (1976 a, 1976 b). The enzyme isolated from Melandrium sp. proved to consist of two subunits (KAMSTEEG et al. 1978). The enzyme described in our work exhibited a HilI number of approximately 2 for cyanidin, indicative for a dimeric configuration of this enzyme as weIl. SALEH et al. (1976 a, 1976 b) reported cyanidin 3-0-glucosyl-transferases in red cabbage and in Haplopappus gracilis. They observed strong inhibition of the enzyme at cyanidin concentrations above 0.25 mM. At lower concentrations the enzyme exhibited Michaelis-Menten type of kinetics. In contrast to these findings we did not observe such extreme inhibition above a critical cyanidin concentration, but only some inhibition at very high concentrations. The data published by SALEH et al. suggest that the extreme inhibition may be caused by impurities of the cyanidin preparation they used and probably not be cyanidin itself. Further studies are needed to explain this discrepancy in substrate inhibition.
464
K. F. F. KHO, J. KAMSTEEG and J. VAN BREDERODE
References BREDERODE, J. VAN, G. VAN NIGTEVECHT, and J. KAMSTEEG: Genetic control and biosynthesis of anthocyanidin-glycosides in the petals of Silene dioica. Heredity 35, 429-430 (1975). BREDERODE, J. VAN and G. VAN NIGTEVECHT: Identification, properties and genetic control of UDP-glucose: isovitexin-7-0-glucosyltransferase isolated from petals of Melandrium album. Mol. Gen. Genet. 122,215-229 (1973). BOURDILLON, C., J. N. BARBOTIN, and D. THOMAs: Kinetic studies of an allosteric enzyme under artificial conformational constrainst imposed by a tight immobilization into proteic membranes. FEBS lett. 68, 27-30 (1976). FLORINI, J. R. and C. S. VESTLING: Graphical determination of the dissociation constants for two-substrate enzyme systems. Biochim. Biophys. Acta (Amst.) 25, 575-578 (1957). FRITSCH, H. and H. GRISEBACH: Biosynthesis of cyanidin in cell cultures of Haplopappus gracilis. Phytochem. 14,2437-2442 (1975). JURD, L.: The acid catalyzed conversion of 3-hydroxyflavanones to anthocyanidins. Phytoehern. 8, 2421-2423 (1969). KAMsTEEG, J., J. VAN BREDERODE and G. VAN NIGTEVECHT: Identification and properties of UDP-glucose: Cyanidin 3LO-glucosyltransferase isolated from petals of the Red Campion (Silene dioica). Bioehern. Genet. 16 (1978). KHO, K. F. F., H. WIERING, and G. J. H. BENNINK: Anthocyanin synthesis in a white flowering mutant of Petunia hybrida. Planta (Ber!.) 127, 271-279 (1975). KHO, K. F. F., A. C. BOLSMAN-LouWEN, J. C. VUIK, and G. J. H. BENNINK: Anthocyanin synthesis in a white flowering mutant of Petunia hybrida. 11. Accumulation of dihydroflavonol intermediates in white flowering mutants; uptake of intermediates in isolated corollas and conversion into anthocyanins. Planta (Ber!.) 135,109-118 (1977). KRISHNAMURTY, H. G., V. KRISHNAMOORTY, and T. R. SESHADRI: Preparation of anthocyanidins and their glycosides from related flavonoids. Phytochem. 2, 47-50 (1963). LINEWEAVER, H., and D. BURK: The determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56, 658-666 (1934). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol re agent. J. Bio!. Chem. 193, 256-275 (1975). PATSCHKE, L. and H. GRISEBACH: Dihydrokaempferol and dihydroquercetin as precursors of kaempferol and quercetin in Pisum sativum. Phytochem. 7,235-237 (1968). RIBEREAU-GAYON, P.: Plant phenolics, p. 137, Oliver and Boyd, Edinburgh, 1972. SALEH, N. A. M., J. E. POULTON, and H. GRISEBACH: UDP-glucose: cyanidin 3-0-glucosyltransferase from red cabbage seedlings. Phytochem. 15, 1865-1868 (1976). SALEH, N. A. M., H. FRITSCH, P. WITKOP, and H. GRISEBACH: UDP-glucose: cyanidin 3-0-glucosyltransferase from cell cultures of Haplopappus gracilis. Planta (Ber!.) 133, 41-45 (1976). SEGEL, I. H.: Enzyme kinetics, p. 346-385, Wiley Interscience, New York, 1974 a. Ibid. p. 100-161 (1974 b). STICKLAND, R. G. and B. J. HARRISON: Precursors and genetic control of pigmentation: I. Induced biosynthesis of pelargonidin, cyanidin and de1phinidin in Antirrhinum maju's. Heredity 33,108-112 (1974). WIERING, H.: Genetics of flower colour in Petunia hybrida HORT. Genen Phaenen 17, 117-134 (1974). WIERING, H. and P. DE VLAMING: Glycosylation and methylation patterns of anthocyan ins in Petunia hybrida. I. The gene Gf. Genen Phaenen 16, 35-50 (1973). Dr. J. VAN BREDERODE, Department of Population and Evolutionary Biology, University of Utrecht, Padualaan 8, Utrecht, The Netherlands.
Z. P/lanzenphysiol. Bd. 88. S. 449-464. 1978.
Identification, Properties and Genetic Control of UDP-Glucose : Cyanidin 3-0-Glucosyltransferase in Petunia hybrida K. F. F.
KHO,
J. KAMSTEEG and J. VAN BREDERODE
With 15 figures Received 27 February 1978 . Accepted 11 March 1978
Summary In corollas of Petunia hybrida the presence of a glucosyltransferase capable of catalyzing the transfer of glucose from UDP-glucose to the 3-0-position of cyanidin was demonstrated. The enzyme could also use delphinidin as a substrate. The glucosyltransferase exhibited a pH optimum between 7.5-8.0 and had a «true Km» value of 1.7 mM for UDP-glucose. For the substrates cyanidin and delphinidin allosteric behaviour was observed. The Hili number was determined to be 1.9 for cyanidin. The K' values were 2.5 X 10-3 mM for cyanidin and 5 X 10-3 mM for delphinidin. Cyanidin 3-glucosylation was inhibited by delphinidin and delphinidin 3-glucosylation was inhibited by cyanidin, either inhibition being of the uncompetitive type. The enzymatic activity was controlled by the genes An1 and An2. Genotypes in which either of these genes was in the homozygous recessive state showed 20 0/0 of the activity of the control value, without changes in the substrate affinities of the enzyme.
Key words: glucosyltransjerase, cyanidin, genetic control, Petunia hybrida.
Introduction Genetic studies of flower colour in Petunia hybrida demonstrated that several genes are involved in the regulation of flavonoid biosynthesis (WIERING, 1974). The synthesis of anthocyanins is governed by the complementary genes An1, An2, and An3. Dominant alleles of each of these genes are required for normal synthesis of cyanidin 3-glucoside in the flower limb. It has been demonstrated that white flowers of the genotype AnlAnlAn2An2an3an3 are capable to convert exogeneous supplied dihydroquercetin into cyanidin 3-glucoside. The genotypes anlanlAn2An2An3An3 and AnlAn1an2an2An3An3 are unable to do this. Moreover, the latter genotypes accumulate dihydroquercetin and related dihydroflavonols in the flower (Figure 1, cf. also KHO et al., 1975, 1977). Obviously, dominant alleles of Anl and An2 are required for the conversion of
z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
450
K.
F. F.
KHO,
J.
KAMSTEEG
J.
and
VAN BREDERODE
r----------, 1
An1, An 2
I OH
OH
1
0
1HO
OH
1
An3
OH
0
--I,\~
I
1 1
1 HO
1
(\1
OH
1 OW
UDPG
~
UDPI
L ________ ....l dihydroflavonoL intermediates
cyanidin
cyanidin - 3 -gLucoside
Fig. 1: Genetic control of cyanidin 3-glucoside biosynthesis in Petunia hybrida. Dominant alleles of An3 are required for synthesis of dihydroflavonol intermediates. The conversion of dihydroquercetin (R = H) into cyanidin 3-glucoside is governed by the genes Anl and An2. This conversion requires at least two re action steps, probably via cyanidin as intermediate. The glucosylation at position 3 may be catalyzed by a glucosyltransferase.
dihydroquercetin into cyanidin 3-glucoside. Dihydroquercetin is a weIl known precursor of anthocyanins and flavonols (PATSCHKE and GRISEBACH, 1968; FRITSCH, and GRISEBACH, 1975; STICKLAND and HARRISON, 1974). The conversion into cyanidin 3-glucoside requires at least two reaction steps: (i) conversion of the dihydroflavonol into the corresponding anthocyanidin by intramolecular rearrangement and removal of a hydroxyl ion (JURD, 1969) and (ii) a glucosylation at position 3. The correct sequence of these reactions has not yet been established. But there is some evidence that the glucosylation re action is the last step. (c.f. Fig. 1, FRITSCH and GRISEBACH, 1975; RIBEREAU-GAYON, 1972). The present work describes the properties ofUDP-glucose: cyanidin 3-0-glucosyltransferase. The presence of this enzyme has been reported in Silene dioica by VAN BREDERODE (1975) and in red cabbage and Haplopappus gracilis bei SALEH et al. (1976 a, 1976 b). The enzyme is responsible for the 3-glucosylation reaction outlined in Fig. 1. and we undertook experiments to deterrriine whether the enzyme is controlled by the genes An1 or An2. Materials and Methods Plant Material The red flowering Petunia line R3 (genotype AnlAnlAn2An2An3An3) and the white flowering mutants W39 (genotype AnlAnlAn2An2an3an3), W38 and W19 (both with genotype anlanlAn2An2An3An3) and W12 and A10 (both with the genotype AnlAnlan2an2An3An3) were cultivated in the greenhouse of the Institute of Genetics, University of Amsterdam. Chemieals UDP-(U-14C)-glucose, 233 Ci/mole and ADP-(U 14C)-glucose, 283 Ci/mole were supplied by the Radiochemical Centre, Amersham, England. UDP-glucose and ADP-glucose were Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
451
purchased from Sigma. The specific activity of the nucleotide sugars was adjusted by addition of carrier to ca. 1 Ci/mole. For determination of specific activity and chemical purity see VAN BREDERODE and VAN NIGTEVECHT (1973'). Cyanidin chloride was synthesized according to KRISHNAMURTY et al. (1962) and purified by preparative chromatography on Whatman-3 paper in the solvent systems butanol-1 acetic acid - water (4: 1 : 5, viviv, upper phase) and 10f0 w/v HCI. Finally, the anthocyanidin was precipitated as an amorphous solid with diethylether. Delphinidin chloride was obtained from Carl Roth A.G., Karlsruhe, Federal Republic of Germany. The 3-glucosides of cyanidin and delphinidin were isolated from appropriate genotypes of Petunia hybrida (WIE RING and DE VLAMING, 1973). Pro tein Assay
Protein was determined according to the method of LOWRY et al. (1951) using bovine albumin as a standard. Enzyme Preparation
All steps were performed between 0 and 4°C. From flower buds of stage III (KHO et al., 1975) the limb part was dissected. One gram of the flower limb material was homogenized in an all glas Pott er homogenizer in 5 ml 50 mM sodium-potassium-phosphate buffer pH 7.5, containing 5 0/0 soluble polyvinylpyrrolidone and 20 mM ß-mercaptoethanol. The homogenate was centrifuged for 10 min at 38,000 X g. To remove endogenous substrate, phenolic substances, soluble PVP and low molecular weight materials, the supernatant was passed through a polyclar AT column (20 X 1 cm) (KAMSTEEG and VAN BREDERODE, 1978) and subsequently through a Sephadex G-50 column (30 X 2.5 cm). The polyclar AT and Sephadex G-50 columns were equilibrated and eluated with 10 mM sodium-potassium phosphate buffer pH 7.5, containing 4 mM ß-mercaptoethanol. Unless noted otherwise the high molecular weight fraction of the Sephadex G-50 eluate was used as an enzyme source. Enzyme Assays
The standard re action mixture consisted of 50 ,ul enzyme, 5 ,ul 10 mM cyanidin, dissolved in 5 mM HCl and 15,u1 10 mM UDP-glucose, labelIed uniformly in the glucose moiety (S.A. ca. 1 Ci/mole). The reaction mixture was incubated for 5 min at 30°C, unless specified otherwise. The re action was stopped by addition of an equal volume of 1 % (w/v) trichloroacetic acid (TCA) in methanol. After addition of carrier cyanidin 3-glucoside, the re action mixture was transferred quantitatively as a spot on Whatman-3 paper and developed two dimensionally in the solvent systems butanol-1-acetic acid water (4 : 1 : 5, v/v/v, upper phase) and 1 % (w/v) HCI respectively. After drying, the spot corresponding to cyanidin 3-glucoside was cut out and placed in a scintillation vial. After addition of scintillation solvent, composed of 4 g 2,5-diphenyloxazole and 50 mg 1,4-bis-2-(5-phenyloxazoyl)-benzene in 1 I toluene, the vial was counted in a Packard liquid scintillation spectrometer (counting yield approximately 80 %). For the determination of zero time control, TCA was added to the reaction mixture before incubation. UDP-glucose : delphinidin 3-0-glucosyltransferase activity was determined by the same procedure as described for cyanidin, using delphinidin as substrate and delphinidin 3-glucoside as a carrier. In some cases cyanidin and delphinidin were tested simultaneously. In this case incorporation of radioactivity in cyanidin 3-glucoside and delphinidin 3-glucoside was measured by addition of both carriers to the reaction mixture and determination of radioactivity in the spots of the 3-glucosides after two dimensional chromatography. Radioactivity found in case of zero time controls was between 0-10 dpm above the background (25-30 dpm) found with an untreated piece of Whatman-3 paper. All enzyme tests were run in duplicate or triplicate. Z. Pjlanzenphysiol. Bä. 88. S. 449-464. 1978.
452
K. F. F.
KHO,
J.
KAMSTEEG
and
J.
VAN BREDERODE
Results
1. Enzymatic Synthesis
0/ cyanidin 3-glucoside
Incubation of UDP-glucose, labelled uniformly in the glucose moiety, with cyanidin and an enzyme preparation from flower buds of the red flowering line R3 - in which the An genes are dominant - resulted in incorporation of radioactivity in cyanidin 3-glucoside. When the enzyme was omitted in the test, or TCA was added before incubation, no incorporation was observed (Table 1). Table 1: Synthesis of cyanidin 3-glucoside from cyanidin and UDP-glucose, catalyzed by an enzyme preparation from flower buds of Petunia hybrida. enzyme fraction
substrate added
additives
dpm
38,000 Xg supernatant
cyanidin none cyanidin cyanidin
none none 0.1 % tween 80 15mM EDTA
3308 1431 2918 1512
polyc1ar AT + sephadex G 50 elutae
cyanidin none cyanidin cyanidin
none none 6 mM MgCl 2 6mM MnCl 2
1568 32 1215 612
none
cyanidin
phosphate buffer pH 7.5
23
polyc1ar AT + Sephadex G 50 eluate
cyanidin
50 ,ul 1 Ofo TCA in methanol
26
Activity was expressed as dpm incorporated in cyanidin 3-glucoside. The nuc1eotide sugar was labelIed with a specific activity of ca. 1 Ci/mole. Enzyme preparations were obtained from the red flowering line R3. The enzyme tests were carried out as described in Materials and Methods.
When cyanidin was omitted in the test of the supernatant of the crude homogenate, about half of the product formation remained, indicating that the supernatant contained endogenous cyanidin. When the supernatant was passed through polyclar AT and Sephadex G-50 columns subsequently, only the high molecular weight fraction proved to be active in the enzyme test. Omitting of cyanidin in this case gave no activity, indicating that the endogenous cyanidin was completely removed. Addition of detergent (Tween 80, 0.1 Ofo final concentration) did not enhance the enzymatic activity. The product formation was reduced by EDTA, but could not be stimulated by divalent metal ions, such as Mg 2+ and Mn 2+ (Table 1). The partial purification of the enzyme is further outlined in Table 2. This Table shows that considerable activity was left in the pellet, which could not be washed out with buffer.
z. Pflanzenphysiol.
Bd. 88. S. 449-464. 1978.
453
UDP-glucose : cyanidin 3-0-glucosyltransferase
Table 2: Purification of UDP-glucose: cyanidin 3-0-glucosyltransferase isolated from the corolla of Petunia hybrida. fraction
protein (mg)
units
crude homogenate
48
119
2.5
54
38,000 Xg pellet rinsed with buHer
22
79
3.6
36
38,000 Xg supernatant
16
219
14
100
92
42
42
Polyclar AT + Sephadex G-50 eluate
2.2
specific activity
recovery
enzyme purification
0/0
5.6 17
One enzyme unit is defined as the amount of enzyme which catalyzes the formation of 1 nmole product per minute at 30 oe in the enzyme assay.
The partial purification of the enzyme is further outlined in Table 2. The specificity of the enzyme test for cyanidin 3-glucoside formation was checked previously by KAMSTEEG and VAN BREDERODE (1978). The quantity of cyanidin 3-glucoside formed from cyanidin and UDP-glucose was proportional with time up to 10 min (Figure 2). The leveling off of the curve after 10 min is probably caused by inactivation of the cyanidin at the pH used. Preincubation of the enzyme for 30 min with UDP-glucose and stopping the re action 5 min after the addition of cyanidin resulted in an equal amount of product forn;'ation as when both substrates were added simultaneously. N
I~
15
x
E
Q.
Fig. 2: Formation of cyanidin 3-glucoside from cyanidin and UDP-glucose as a function of time. The incubations were carried out under standard conditions as described in Materials and Methods.
o
2 5
10
20
30 minutes
Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
454
K.
F. F.
KHO, }. KAMSTEEG
and J.
The product formation was found enzyme (Figure 3).
to
VAN BREDERODE
be proportional to the amount of added
E 1200
0.
"
800
400
o
10
20
30
40
50
Jll
enzyme
Fig. 3: Formation of cyanidin 3-glucoside from cyanidin and UDP-glucose as a function of added enzyme. The enzyme was assayed as described in Materials and Methods. The pH optimum was determined to be in the range 7.5-8.0 (Figure 4). In connection with the pH range of the phosphate buffer further tests were run at pH 7.5. N
'~ x
15
E
J
a.
"t:>
10
5
5
6
7
B
9
pH
Fig. 4: Effect of pH on UDP-glucose: cyanidin 3-0-glucosyltransferase. The reaction mixture contained in a total volume of 107 .u1: 50 .ug protein, 12.5 ,umoies potassiumsodium phosphate, 200 nmoles ß-mercaptoethanol, 240 nmoles UDP-(U14C)-glucose (S. A., 1 Ci/mole), 83 nmoles cyanidin. Incubation mixtures prepared in parallel (cyanidin and UDP-glucose omitted) were used to determine pH.
The results indicate clearly the presence of an enzyme, capable of catalyzing the transfer of the glucose moiety from UDP-glucose to the 3-0-position of cyanidin in flower limbs of Petunia hybrida.
2. Substrate Specificity The enzyme was also capable to catalyze the transfer of the glucose moiety from UDP-glucose to the 3-0-position of delphinidin (Table 3). Neither for cyanidin, nor for delphinidin ADP-glucose could serve as a glucose donor. For delphinidin glucosylation a similar pH optimum as mentioned for cyanidin (7.5-8.0) was observed.
z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
455
Table 3: Substrate specificity of UDP-glucose : cyanidin 3-0-glucosyltransferase of Petunia hybrida. Sugar donor
acceptor
carrier
radioactivity cochromatographed with the carrier (dpm)
UDP-glucose
cyanidin
2885
UDP-glucose
delphinidin
ADP-glucose
cyanidin
ADP-glucose
delphinidin
cyanidin 3-glucoside delphinidin 3-glucoside cyanidin 3-glucoside delphinidin 3-g1ucoside
2619
3 5
The enzyme was isolated from genotype R3 and purified by filtration over polyclar AT and Sephadex G-50 columns. The re action mixture contained 500 nmoles potassium-sodium phosphate buHer, pH 7.5, 200 nmoles ß-mercaptoethanol, 50 nmoles of UDP-(U 14C)glucose (S.A. 6 Ci/mole) or ADP-(U-14C)-glucose (S.A. 6 Ci/mole), 50 nmoles of the anthocyanidin and 16 flg protein. Radioactivity was determined in the spot of the carrier substance after cochromatography with the re action mixture as described in Materials and Methods and given as dpm above the background (25-30 dpm).
3. Glucosyltransferase Activity in Various Stages of Flower Bud Development In the vegetative parts of the plant no enzymatic activity could be detected. Figure 5 shows that the glucosyltransferase activity started to develop after initiation of the flower bud and could be detected in the corolla. The highest activity was present in bud stage III, according to KHO et al. (1975). This stage is correlated with rapid ..
~
150
...
Cl
I
.~ 100
Fig. 5: Activity of the glucosyltransferase in various stages of the developing flower budo For activity measurements 38,000 X g supernatants were used. Assays were carried out as described in Materials and Methods. Bud length is defined as the distance between the flower receptacle and the top of the corolla (KHO et al., 1975). Stage I: 0-5 Olm, Stage 11: 5-15 Olm, Stage 111: 15-35 Olm, Stage IV, 35 mmopen flower, Stage V: open flower, G: vegetative material.
vi
GI
"0 E I:
50
o...L..=._'::l-n-':='---':::I-...I....L.--Ln~ G I :n ][ :nz: J[ bud stage
z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
456
K.
F. F.
KHO,
J.
KAMSTEEG
and }.
VAN BREDERODE
elongation of the tubular corolla and with a high rate of anthocyanin synthesis. During later stages of flower life the glucosyltransferase activity decreased markedly. 4. Kinetics LINEWEAVER and BURK (1934) plots of the reciprocal of the initial velocity of the cyanidin glucosylation reaction versus the reciprocal of the UDP-glucose concentration at different fixed cyanidin concentrations show that the relationship between V-i and [UDP-glucose]-i is virtually linear, the apparent Km value being dependent upon the second substrate (cyanidin) concentration (Figure 6).
v- 1
30
1.0
2.0
3.0
[UDPGr 1 mM- 1
Fig. 6: Lineweaver & Burk plot of the reciprocal of the initial velocity of the cyanidin 3glucosylation re action versus reciprocal of mMolar concentration of UDP-glucose. The following concentrations of cyanidin were used: 0.0308 mM (0--0); 0.0431 mM (e-e); 0.0631 mM (0-0); 0.1369 mM ( . - . ) . The reaction mixture contained in a total volume of 65,u1: 500 nmoles potassium-sodium phosphate buffer pH 7.5, 200 nmoles ßmercaptoethanol, 8,ug protein, the indicated amount of cyanidin and UDP-glucose in a concentration ranging from 0.36 to 0.89 mM.
When the reciprocal of the initial velocity was plotted versus the reciprocal of the cyanidin concentration at different fixed UDP-glucose concentrations, no linear relationship between V-i and [cyanidin]-i was observed. However, when the reciprocal of the initial velocity was plotted versus the reciprocal of the square of the cyanidin concentration, a virtually linear relationship was obtained (Figure 7). These kinetic responses indicate that the enzyme behaves as a Michaelis-Menten system with respect to UDP-glucose, but as an allosteric system (SEGEL, 1974 a) with respect to the cyanidin substrate. A secondary plot according to FLORINI and VESTLING (1951) of the intercepts on the V-i axis of Figure 6 versus the reciprocals of the square of the fixed cyanidin concentrations yielded the Vmax and the K' value for cyanidin. A secondary plot of the intercepts on the V-i axis obtained from Figure 7 versus the reciprocals of the fixed UDP-glucose concentration yielded the «true Km» value for
z.
Pflanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
~ I
457
20
E
Co
"C
400
800
1200
[CVANIDINr 2
mM- 2
Fig. 7: Plot of the reciprocal of initial velocity of the cyanidin 3-glucosylation reaction versus reciprocal of the square of mM concentration of cyanidin. The following concentrations of UDP-glucose were used: 0.36 mM (0-0); 0.45 mM (e-e); 0.54 mM (0--0); 0.89 mM ( . - - . ) . The reaction mixture contained in a total volume of 65.ul: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles p-mercaptoethanol, 8.ug protein, the indicated amount of UDP-glucose and cyanidin in a concentration ranging from 0.0308 to 0.1369 mM.
-1 V max 10 I
E
Co
"C
Km -1 1.0 I
800 (K,)-1
mM-1
[UDPG]-1 0 I
400
I
0
2.0
1.0 I
400
I
800
[CVANIDINr 2
3.0 I
1200 mM- 2
Fig. 8: Plot of the intercepts on the vertical axis of the data plotted in Figures 6 and 7 (corresponding to lIvmax in the presence of the second substrate as the limiting factor), replotted versus reciprocal of corresponding fixed UDP-glucose concentration (0-0) or reciprocal of the square of fixed cyanidin concentration (e-e). The intercept on the vertical axis equals l/vmax and the intercepts on the horizontal axis equal l/true Km for UDP-glucose an.d l/K' for cyanidin. Z. Pjlanzenphysiol. Bd. 88. S, 449-464. 1978.
458
K. F. F. KHO,
J. KAMSTEEG and J. VAN BREDERODE
UDP-glucose. Both secondary plots are given in Figure 8. The kinetic parameters are listed in Table 4. The kinetics of delphinidin 3-0-glucosylation was determined in a similar manner as mentioned for cyanidin 3-0-glucosylation and is presented in the Figures 9, 10 and 11. The relationship between V-i and [delphinidinJ-2 was also virtually linear. The kinetic parameters of this reaction are also listed in Table 4. The kinetic responses for cyanidin indicate a Hill number (SEGEL, 1974 a) of the enzyme elose to 2.0 for anthocyanidin substrates and elose to 1.0 for UDP-glucose. Direct determination of the HilI number by HilI plots (BOURDILLON et al., 1976) Table 4: Kinetic parameters for enzymatic transfer of glucose from UDP-glucose to the 3-0 position of cyanidin or delphinidin by a purified enzyme preparation from flower buds of the red flowering line R3 of Petunia hybrida. The data were obtained from the plots given in the figures 8 and 11. K'b) delphinidin (mM)
reaction
V max (nmoies . min- 1 . mg protein-1 )
Kma) UDP-glucose (mM)
K'b) cyanidin (mM)
cyanidin 3-glucosylation delphinidin 3-glucosylation
69
1.70
2.5 X 10-3
54
1.02
5.0 X 10-3
a) «True» Michaelis constant. b) K' is defined as the substrate concentration at ~ Vmax raised Hill number (cf. SEGEL, 1974 a).
to
apower equal to the
v- 1 10
1.0
2.0 [UPDGr 1
3.0 mM- 1
Fig. 9: Lineweaver & Burk plot of the initial velocity of the delphinidin 3-glucosylation re action versus reciprocal of mM concentration of UDP-glucose at fixed concentrations of delphinidin. The following delphinidin concentrations were used: 0.0383 mM (e-e); 0.0585 mM (0-0); 0.0802 mM ( . - . ) ; 0.1185 mM (0-0). The reaction mixture contained in a total volume of 65,u1: 500 nmoles potassium-sodium phosphate buffer, pH 7.5, 200 nmoles ß-mercaptoethanol, 13.5,ug protein, the indicated amount of delphinidin and UDP-glucose in a concentration ranging from 0.374-0.892 mM. Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
200
400
600
[DELPHINIDINr 2
459
BOO
mM- 2
Fig. 10: Plot of the recipocal of initial re action velocity of delphinidin 3-glucosylation versus reciprocal of the square of mM concentration of delphinidin at fixed concentrations of UDP-glucose. The following UDP-glucose concentrations were used: 0.374 mM (e---e); 0.552 mM (0-0); 0.892 mM (0-0). The reaction mixture contained in a total volume of 651ll: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles ß-mercaptoethanol, 13.5 flg protein, the indicated amount of UDP-glucose and delphinidin in a concentration ranging from 0.0383'---0.1185 mM. V -1 max 10
'I E Co
...."tJ ~
5
Km -1
[UDPG]-1 mM- 1 1.0
0 I
300 200
(KT 1
I
100
I
0
2.0
1.0 I
100 200
I
300
I
I
3.0 I
400 500 600
I
700
[DELPHINIDI N] - 2 mM- 2
Fig. 11: Plot of the intercepts on the vertical axis of the data plotted in Figures 9 and 10 (corresponding to l/vmax in the presence of the second substrate as the limiting factor) replotted versus reciprocal of corresponding fixed UDP-glucose concentration (0---0) or reciprocal of the square of corresponding fixed delphinidin concentration (e---e). The intercept on the vertical axis equals l/vmax and the intercepts on the horizontal axis equal lItrue Km for UDP-glucose and lIK' for delphinidin.
proved to be unsatisfactory (TABAK, personal communication), because V max cannot be determined directly, as the enzyme tends to be inhibited to some extent at high anthocyanidin concentrations. Therefore we determined the Hill number by another method. We calculated the correlation coefficient for linear regression between V-i and [substrate]-n in case of cyanidin and UDP-glucose. The value of n that gives the highest correlation coefficient is equivalent to the Hill number (SEGEL, Z. Pflanzenphysiol. Bd. 88. S. 449-464. 1978.
460
c.
K.
F. F.
KHO,
J.
KAMSTEEG
and
J.
VAN BREDERODE
lOOO
'(3
:::.. o
u
.:5
:;;
0.990
~o u
0.980
(e_)
0.970
o
Fig. 12: Correlation coefficients for linear regression between v-1 and [substrate concentration]-n as a function of n for cyanidin and for UDP-glucose (0--0). The value of n corresponding to the highest correlation coefficient equals the Hill number for the substrate.
lO
1.5 hill
2.0
2.5
number
1974 a). As can be seen in Figure 12, the value of the Hill number was dose to 1.0 for UDP-glucose and dose to 2.0 (1.9) for cyanidin.
5. Competition Experiments involving Simultaneous Reaction of Cyanidin and Delphinidin with UDP-glucose Our test system allows investigation of the influence of delphinidin on the 3-0-glucosylation re action of cyanidin and vice versa. When both cyanidin and delphinidin were incubated with UDP-(UßC)-glucose in the presence of the enzyme preparation from genotype R3 and the reaction mixture was chromatographed two-dimensionally together with the carrier substances cyanidin 3-glucoside and delphinidin 3-glucoside, radioactivity was incorporated in the spots corresponding to cyanidin 3-glucoside and delphinidin 3-glucoside. Plots of the reciprocal of the initial velocity versus the reciprocal of the square of the cyanidin concentration at various fixed delphinidin concentrations are given in Figure 13. The corresponding plots for delphinidin at various fixed cyanidin concentrations are given in Figure 14. The graphs indicate that the 3-0-glucosylation of cyanidin is uncompetitively inhibited by delphinidin and vice versa (SEGEL, 1974 b). At high concentrations of both substrates, the sum of both reaction velocities was virtually constant. 6. Genetic Control
The activity of the glucosyltransferase in flower limb extracts of various genotypes of Petunia hybrida is presented in Figure 15. In the white flowering mutant W39 (genotype An1An1An2An2an3an3) the enzymatic activity proved to be in the same order as in the red flowering line R3 (genotype An1An1An2An2An3An3), which was used as a contro!. This indicates that the gene An3 is not involved in regulation of the glucosyltransferase activity, but controls probably another step in anthocyanin biosynthesis (cf. Figure 1). Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
461
v- 1 ..
E
...
20
Q.
500 [CVANIDIN]-2
1000 mM-2
Fig. 13: Plot of the reciprocal of initial reaction velocity of cyanidin 3-glucosylation versus reciprocal of the square of cyanidin concentration at fixed delphinidin concentrations: 0 mM 0.032 mM (0-0); 0.100 mM (0-0). The reaction mixture contained in a total volume of 75,ul: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles ji'-mercaptoethanol, 13.5,ug protein, 116 nmoles UDP-glucose, the indicated amount of delphinidin and cyanidin in a concentration ranging from 0.0397 to 0.0891 mM.
v- 1 ,. 10 E Q.
...
100
300
500
[DELPHINIDINr 2 mM-2
Fig. 14: Plot of the reciprocal of initial velocity of delphinidin 3-glucosylation versus reciprocal of the square of delphinidin concentration at fixed concentrations of cyanidin: o mM 0.025 mM (0--0); 0.077 mM (0-0). The re action mixture contained in a total volume of 75,ul: 500 nmoles potassium-sodium phosphate buHer pH 7.5, 200 nmoles ji'-mercaptoethanol, 13.5,ug protein, 116 nmoles UDP-glucose, the indicated amount of cyanidin and delphinidin in a concentration ranging from 0.0492 to 0.1154 mM.
Genotypes with either An1 or An2 heing homozygous recessive showed a remarkahly lower enzymatic activity, not exceeding 20 Ofo of the line R3, used as a contro!. The mutants W19 and W38 are homozygous recessive for the gene An1 hut are different with regard to other genes, not related to the investigated part of the
z. Pjlanzenphysiol. Bd. 88. S. 449--464. 1978.
462
K. F. F.
KHO,
J.
KAMSTEEG
and
J.
VAN BREDERODE
'j'
-.
~ 20 CI
'j' c:
oe
ui
10
GI
-0
E c: R3
W 39
Fig. 15: Genetic control of glucosyl transferase activity in flower buds of Petunia hybrida. Activities were determined in 38,000 X g supernatants as described in Materials and Methods for cyanidin 3-0-glucosylation ( D ) and delphinidin-3-0-glucosylation (~ ). The following lines were investigated: R3 (genotype AnlAnlAn2An2An3An3); W39 (genotype AnlAn1An2An2an3an3); W38 (genotype an1anlAn2An2An3An3); W 19 (genotype anlanlAn2An2An3An3); W12 (genotype AnlAnlan2an2An3An3); AIO (genotype Anl Anl an2an2An3An3).
biosynthetic pathway. The genotypes W12 and AlO are homozygous recessive for the gene An2 and differ also in their genetic background (WIERING and DE VLAMING, unpublished results). The portein content in these genotypes was not seriously lowered and the specific activity of the enzyme did not exceed 30 % of the control value, estimated for R3. Kinetic studies revealed no significant differences of the substrate affinities for the enzymes present in genotypes homozygous recessive for either Anl or An2 or the control genotype R3, in which both genes are dominant. The results demonstrate a regulatory role of both genes Anl and An2 upon the total amount of the glucosyltransferase present in the flower limb of Petunia. Discussion Our results demonstrate clearly that the ultimate step of the biosynthetic route leading to cyanidin 3-glucoside has to be the glucosylation of cyanidin at position 3 in Petunia hybrida in agreement with the scheme postulated in Figure 1. An enzyme, capable of catalyzing this reaction, involving the transfer of a glucosyl residue from UDP-glucose to the 3-0-position of cyanidin has been detected in developing flower buds of Petunia hybrida. In vegetative plant material no such enzymatic activity could be detected. Th~ enzyme becomes detectable during the later stages of flower bud development, indicating that it is synthesized as a result of differential gene activity concerning flower development. The enzyme preparations were also capable to transfer glucose from UDP-glucose to delphinidin. However, in the genotypes investigated, cyanidin 3-glucoside is the major anthocyanin present. The partially purified enzyme preparations exhibited both for the substrates Z. Pjlanzenphysiol. Bd. 88. S. 449-464. 1978.
UDP-glucose : cyanidin 3-0-glucosyltransferase
463
cyanidin and delphinidin allosteric properties (SEGEL, 1974 a), and we determined the average HilI number to be 1.9. For the second substrate (UDP-glucose) we observed the common Michaelis-Menten type of kinetics (nh = 1.0). The kinetic constants (K') differ only slightly for cyanidin or delphinidin. As cyanidin is the normal substrate in our genotypes we designated the enzyme as a glucosyltransferase for cyanidin. The allosteric behaviour of the enzyme with regard to anthocyanidin substrates may be functional, since anthocyanidins are slightly soluble in aqueous environment and are probably produced on membranes (FRITSCH and GRISEBACH, 1975). The activation of the enzyme by a second binding site may be essential to achieve dissociation of the anthocyanidin from the membrane and simultaneous bin ding to the soluble enzyme. The soluble UDP-glucose however is randomly present in the environment of the enzyme and requires probably no special reaction mechanism for sufficient reaction rates. Thus, a Michaelis-Menten type reaction will ensure sufficient participation of UDP-glucose in the reaction. Competition experiments revealed that delphinidin inhibited the glucosylation of cyanidin and vice versa, but the sum of both activities was virtually constant. The inhibition resembles the uncompetitive model (SEGEL, 1974 b), suggesting that one enzyme is able to catalyze the 3-glucosylation of both anthocyanidins. Further evidence for this may be derived from the virtually constant ratio of both enzymatic activities in various genotypes and from the equal pH optima. The genes An1 and An2 are both involved in the regulation of the glucosyltransferase activity. When one of these genes is homozygous recessive, the enzyme activity is reduced to 20-30 Ofo of the original value, without significant differences of kinetic parameters. A genotype in which both genes are homozygous recessive could not be tested, because it was not available. The observed data indicate that both genes play a regulatory role with regard to the amount of glucosyltransferase activity present and are not to be considered as structural genes, controlling this activity. UDP-glucose: cyanidin-3-0-glucosyltransferases have been reported earlier by VAN BREDERODE et al. (1975) and by SALEH et al. (1976 a, 1976 b). The enzyme isolated from Melandrium sp. proved to consist of two subunits (KAMSTEEG et al. 1978). The enzyme described in our work exhibited a HilI number of approximately 2 for cyanidin, indicative for a dimeric configuration of this enzyme as weIl. SALEH et al. (1976 a, 1976 b) reported cyanidin 3-0-glucosyl-transferases in red cabbage and in Haplopappus gracilis. They observed strong inhibition of the enzyme at cyanidin concentrations above 0.25 mM. At lower concentrations the enzyme exhibited Michaelis-Menten type of kinetics. In contrast to these findings we did not observe such extreme inhibition above a critical cyanidin concentration, but only some inhibition at very high concentrations. The data published by SALEH et al. suggest that the extreme inhibition may be caused by impurities of the cyanidin preparation they used and probably not be cyanidin itself. Further studies are needed to explain this discrepancy in substrate inhibition.
464
K. F. F. KHO, J. KAMSTEEG and J. VAN BREDERODE
References BREDERODE, J. VAN, G. VAN NIGTEVECHT, and J. KAMSTEEG: Genetic control and biosynthesis of anthocyanidin-glycosides in the petals of Silene dioica. Heredity 35, 429-430 (1975). BREDERODE, J. VAN and G. VAN NIGTEVECHT: Identification, properties and genetic control of UDP-glucose: isovitexin-7-0-glucosyltransferase isolated from petals of Melandrium album. Mol. Gen. Genet. 122,215-229 (1973). BOURDILLON, C., J. N. BARBOTIN, and D. THOMAs: Kinetic studies of an allosteric enzyme under artificial conformational constrainst imposed by a tight immobilization into proteic membranes. FEBS lett. 68, 27-30 (1976). FLORINI, J. R. and C. S. VESTLING: Graphical determination of the dissociation constants for two-substrate enzyme systems. Biochim. Biophys. Acta (Amst.) 25, 575-578 (1957). FRITSCH, H. and H. GRISEBACH: Biosynthesis of cyanidin in cell cultures of Haplopappus gracilis. Phytochem. 14,2437-2442 (1975). JURD, L.: The acid catalyzed conversion of 3-hydroxyflavanones to anthocyanidins. Phytoehern. 8, 2421-2423 (1969). KAMsTEEG, J., J. VAN BREDERODE and G. VAN NIGTEVECHT: Identification and properties of UDP-glucose: Cyanidin 3LO-glucosyltransferase isolated from petals of the Red Campion (Silene dioica). Bioehern. Genet. 16 (1978). KHO, K. F. F., H. WIERING, and G. J. H. BENNINK: Anthocyanin synthesis in a white flowering mutant of Petunia hybrida. Planta (Ber!.) 127, 271-279 (1975). KHO, K. F. F., A. C. BOLSMAN-LouWEN, J. C. VUIK, and G. J. H. BENNINK: Anthocyanin synthesis in a white flowering mutant of Petunia hybrida. 11. Accumulation of dihydroflavonol intermediates in white flowering mutants; uptake of intermediates in isolated corollas and conversion into anthocyanins. Planta (Ber!.) 135,109-118 (1977). KRISHNAMURTY, H. G., V. KRISHNAMOORTY, and T. R. SESHADRI: Preparation of anthocyanidins and their glycosides from related flavonoids. Phytochem. 2, 47-50 (1963). LINEWEAVER, H., and D. BURK: The determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56, 658-666 (1934). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol re agent. J. Bio!. Chem. 193, 256-275 (1975). PATSCHKE, L. and H. GRISEBACH: Dihydrokaempferol and dihydroquercetin as precursors of kaempferol and quercetin in Pisum sativum. Phytochem. 7,235-237 (1968). RIBEREAU-GAYON, P.: Plant phenolics, p. 137, Oliver and Boyd, Edinburgh, 1972. SALEH, N. A. M., J. E. POULTON, and H. GRISEBACH: UDP-glucose: cyanidin 3-0-glucosyltransferase from red cabbage seedlings. Phytochem. 15, 1865-1868 (1976). SALEH, N. A. M., H. FRITSCH, P. WITKOP, and H. GRISEBACH: UDP-glucose: cyanidin 3-0-glucosyltransferase from cell cultures of Haplopappus gracilis. Planta (Ber!.) 133, 41-45 (1976). SEGEL, I. H.: Enzyme kinetics, p. 346-385, Wiley Interscience, New York, 1974 a. Ibid. p. 100-161 (1974 b). STICKLAND, R. G. and B. J. HARRISON: Precursors and genetic control of pigmentation: I. Induced biosynthesis of pelargonidin, cyanidin and de1phinidin in Antirrhinum maju's. Heredity 33,108-112 (1974). WIERING, H.: Genetics of flower colour in Petunia hybrida HORT. Genen Phaenen 17, 117-134 (1974). WIERING, H. and P. DE VLAMING: Glycosylation and methylation patterns of anthocyan ins in Petunia hybrida. I. The gene Gf. Genen Phaenen 16, 35-50 (1973). Dr. J. VAN BREDERODE, Department of Population and Evolutionary Biology, University of Utrecht, Padualaan 8, Utrecht, The Netherlands.
Z. P/lanzenphysiol. Bd. 88. S. 449-464. 1978.