Planta
Planta (1984) 160:341-347
9 Springer-Verlag 1984
Properties and genetic control of anthocyanin 5-O-glucosyltransferase in flowers of Petunia hybrida L.M.V. Jonsson, M.E.G. Aarsman, J. van Diepen, P. de Vlaming, N. Smit and A.W. Schram Research Section 'Biosynthesis of Flavonoids', Departments of Plant Physiology and Genetics, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, The Netherlands
Abstract. An anthocyanin 5-O-glucosyltransferase from flowers of Petunia hybrida was purified about 30-fold. Using uridine 5'-diphosphoglucose as glucose donor (Km 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (Km 3 gM), isolated from pollen of Petunia. Delphinidin 3-rutinoside, cyanidin 3-rutinoside and delphinidin 3-glucoside did not serve as substrates. The glucosylation of petunidin 3-(p-coumaroyl)-rutinoside showed a pH-activity optimum at pH 8.3 and was neither stimulated by Mg 2 § or Ca 2 § nor inhibited by ethylenediaminetetraacetic acid. After separating the 5-O-glucosyltransferase from the anthocyanidin 3-O-glucosyltransferase by means of chromatofocusing, it was shown that both enzymes exhibit a high degree of positional specificity. The 5-0glucosyltransferase activity was correlated with the gene Anl, but not with the gene Gf. Key words: Anthocyanin biosynthesis - Anthocyanin glucosyltransferase - Anthocyanidin 3-(/)coumaroyl)-rutinoside - Petunia.
Introduction The anthocyanin pigments, widely found in higher plants, occur in vivo as glycosides. The glycosyl groups are bound to one or more of the phenolic hydroxyl groups of the flavonoid C15-skeleton. Until now, 3-glycosides, 3,5-bisglycosides and 3,7bisglycosides have been described (Hahlbrock 1981). All anthocyanins found in flowers of Petunia hybrida are glucosylated at the 3-position. Depending on the genetic background, they may Abbreviations. HPLC=high performance liquid chromatography; 3GT = 3-O-glucosyltransferase; 5GT = 5-O-glucosyltransferase; 3RGac=3-(p-coumaroyl)-rutinoside; 3RGac5G=3-(pcoumaroyl)-rutinoside-5-glucoside; UDPGlc=uridine 5'-diphosphoglucose
also possess a glucose group at the 5-position. Other substitutions of the anthocyanin molecule involve rhamnosylation, acylation by p-coumaric acid and methylation of the B-ring. Using genetic information, the order of these modification reactions has been established (Wiering 1974). The proposed biosynthetic pathway is illustrated in Fig. 1. The glucosylation at the 3-position is carried out by an anthocyanidin 3-O-glucosyltransferase (3 GT) using uridine Y-diphosphoglucose (UDPGIc) as the glucose donor. The genetic control and some properties of this enzyme have been described (Kho et al. 1978). The level of 3GT activity is 5-20% of the normal level when one of the two genes Anl and An2 is homozygous recessive, but residual 3 GT activity was still found in plants homozygous recessive for both these genes (Gerats et al. 1983). It was suggested that an anthocyanin 5-O-glucosyltransferase (5 GT), without an absolute position specificity, might account for this residual activity. The glucosylation at the 5-position is controlled by the gene Gf (Wiering and de Vlaming 1973). Genetic evidence indicates that this pleiotropic gene controls both glycosylation at the 5-position and acylation at the 3-rhamnose group. This situation seems to exist in anthocyanin biosynthesis in Solanaceae in general; genes with effects similar to that of Gfwere found in Solanum tuberosum (Harborne 1960) as well as in Solanum melongena (Tigchelaar et al. 1968). We assume that 5-glucosylation and acylation are controlled by two different genes, and that these steps are carried out in a certain, arbitrary order. The gene Gf is supposed to control the first step, which allows the second to occur.
In order to study the specificity of the anthocyanin glucosyltransferases in Petunia hybrida and to investigate whether Gfcontrols 5-glucosylation, the anthocyanin 5GT was isolated. In this paper we report the results of biochemical studies on the properties and genetic control of the anthocyanin
L.M.V. Jonsson et al. : Anthocyanin 5-O-glucosyltransferase in flowers of Petunia
342
HO--[~O+ ~ ; H OH
grey1
O-GIc
de[phinidin 3-glucoside
.o
-ro>CG.
"OH
grey2
OH O-Gtc-O-Rha delphinidin 3-rutinoside
10f
pu,0,e O-Gtc O- 6tc-O-Rha- p.Cum delphinidin 3-(p-cumQroyl) - rutinosido-5-gtucoside
I M t l Mi2 Mf 1 Mf2
.o-rf-rO
~OCH3
o.
purp,e
~0H O-Gtc
O-Gtc-O-Rha- p.Cum
petunidin 3- (p-cumoroyl}-rutinosido - 5- glucoside
Nil Mr2 Mfl Mf2
jOCH 3 HO" - F ' ~ j ~ k / ' OH purple ~--~ "---~OCH3 O-Gtc O-Gtc-O-Rha-p.Cum molvidin 3-(p-cumoroyl}- rutinosido- 5-glucoside
Fig, 1. Pathways of glycosylation, acylation and methylation reactions in anthocyanin biosynthesis in flowers of Petunia hybrida as deduced from genetic evidence
glucosyltransferases, with special emphasis on 5GT. Materials and methods Plant material. All plants (Petunia hybrida hort.) were grown under greenhouse conditions. The genotypes of the different inbred lines are given in Table 5.
Enzyme extraction. All steps were performed at 0-4 ~ C. Limbs of flowers and flower buds were homogenized with 100 mM potassium phosphate, pH 8.5, containing ~.4 mM fl-mercaptoethanol (5 ml buffer per 25 limbs) and some quartz sand in a mortar. During homogenization, Dowex 1X2-200 (Sigma, St. Louis, Mo., USA) at half the amount of the fresh weight of the tissue was added. For the study of 5GT activity in different mutants, only buds of 15-35 mm length (measured from the base of the corolla) were used. The homogenate was centrifuged at 38,000 g for 20 min. An aliquot of the supernatant liquid (2.5 ml) was chromatographed on a Sephadex G-25 column (1.5 cm diameter, 8.3 cm long; Pharmacia, Uppsala, Sweden), which had been pre-equilibrated with 100 mM potassium phosphate, pH 7.7. The same buffer was used for elution, and fractions containing protein were pooled for assay of 5GT activity and protein content.
Protein was determined according to Bradford (1976) with bovine serum albumin as reference protein.
Molecular weight determination. The molecular weights of glucosyltransferases were determined with crude enzyme preparations after Sephadex G-25 chromatography, using a Sephacryl S-200 Superfine (Pharmacia) column (1.6 cm diameter, 60 cm long) with 100 mM potassium phosphate, pH 7.7, as elution buffer. The column was calibrated with lysozyme (M~ 14,300), trypsinogen (M r 24,000), chymotrypsinogen (M r 25,000), ovalbumin (M r 45,000), bovine serum albumin (M, 66,000) and aldolase (M, 158,000). Chromatofocusing of enzymes. Proteins were precipitated from supernatant liquid (see preceeding section) by ammonimn sulphate at ~ 4 ~ C. The supernatant was brought to 80% saturation by adding solid (NH4)2SO4 over a period of 20 min, during which the pH of the solution was maintained at pH 7.3-7.6 by adding drops of 1 M KOH. The suspension was centrifuged at 38,000 g for 20 min, and the resulting pellet was resuspended in 25 mM histidine-HC1 pH 6.2. After recentrifugation under identical conditions, the final supernatant liquid was applied to a Sephadex G-25 column (pre-equilibrated and eluted with 25 mM histidine-HC1, pH 6.2) to remove residual (NH4)2SO 4. The protein-containing fractions were then applied to a PBE 94 (Pharmacia) chromatofocusing column (0.55cm diameter, 17 cm long), which had been pre-equilibrated with 25 mM histidine-HC1, pH 6.2. The column was eluted with Polybuffer 74 (Pharmacia), pH 4.0, at a flow rate of 35 ml h-1. Fractions of 2.0 ml were collected in tubes containing 100 ~tl 2 M potassium phosphate, pH 8.1. Pooled fractions were concentrated using an ultrafiltration cell (Amicon, Lexington, USA), provided with a PM-10 membrane~ The enzyme preparations used for determining the pH-activity profile were applied to a Sephadex G-25 column and eluted with 10 mM potassium phosphate, pH 7.7. Partial purification of 3-O-glucosyltransferase. The white-flowering mutants W37 and W39 were used as source of enzyme. These lines are homozygous recessive for the gene An3 and blocked in an early step of anthocyanin biosynthesis (Wiering et al. 1979) but contain normal levels of 3GT-activity (Kho et al. 1978). All steps were performed at 0-4 ~ C. For one isolation 25 g (fresh weight) limbs of flowers and flower buds were homogenized in a mortar with some sand and 60 ml 10 mM potassium phosphate buffer pH 7.5, containing 20 m M fl-mercaptoethanol and 5% polyvinylpyrrolidone (w/v). The homogenate was centrifuged at 38,000 g during 20 min. A second extraction of the pellet using 30 ml buffer and re-centrifugation (38,000 g, 20 min) followed. The supernatants were combined and applied to a polyvinylpolypyrrolidone column (12.0 cm long, 5.8 cm diameter), pre-equilibrated and eluted with 10 mM potassium phosphate (pH 7.5). Protein-containing fractions were pooled and dialyzed during 16 h against 10 mM potassium phosphate buffer pH 7.0 and subsequently applied to a DEAEcellulose column (2.4 cm diameter, 6.5 cm long), pre-equilibrated with the same buffer. The bound protein was eluted with 300 ml of a linear gradient of 0 to 1.0 M NaC1 in 10 mM potassium phosphate (pH 7.0). Fractions containing 3GT-activity were pooled and dialyzed against 25 mM histidine-HC1 pH 6.2 and thereafter subjected to chromatofocusing on a PBE 94 column (Pharmacia) as described in the preceeding section. The fractions containing 3GT-activity were pooled and concentrated using an ultrafiltration cell (Amicon), provided with a UM-2 membrane. The enzyme was dialyzed against water and thereafter lyophilized.
Preparation of antiserum. 3 GT was purified as described above. 300 I~g lyophilized enzyme protein dissolved in 1 ml phosphatebuffered saline (pH 7.4) was mixed with 1 ml complete Freunds adjuvant and injected intramuscularly in a rabbit at four differ-
L.M.V. Jonsson et al. : Anthocyanin 5-O-glucosyltransferase in flowers of Petunia ent sites. Three booster injections with the same enzyme preparation, mixed with 1 ml incomplete Freunds adjuvant were given three, five and seven weeks later. In the l l t h week a booster of 600 lag protein was given and in the 12th week the rabbit was plasmaphoresed. A new injection of 250 lag protein, mixed with 1 ml incomplete Freunds adjuvant was given in the 20th week. After two more weeks the rabbit was again plasmaphoresed (50 ml). Complement was inactivated by incubation of the antiserum at 56~ C for 30 min. A crude immunoglobulin fraction was obtained by precipitation with (NH4)2SO 4 to 50% saturation, followed by dialysis against phosphate-buffered saline (pH 7.4).
Incubation of enzyme with antiserum immobilized to Protein ASepharose CL-4B. The coupling of anti-3 GT to Protein A-Sepharose CL-4B (Pharmacia) was carried out at 20 ~ C for 2 h, with gentle rotation in Eppendorf tubes. To each tube containing 7.5 mg Protein A-Sepharose CL-4B was added 0.5, 1.5 or 2.5 mg anti-3GT serum-protein in 250 lal phosphate-buffered saline (pH 7.4). Control incubations contained corresponding amounts of normal rabbit serum. The gel was washed three times with phosphate-buffered saline (pH 7.4) by mixing on a vortex mixer and centrifugation (10 s, 10,000 g). To prevent inhibition of immunoprecipitation caused by Polybuffer, the enzyme samples were applied to a Sephadex G-25 column and eluted with 100 mM potassium phosphate (pH 7.7) before incubation with immobilized antiserum. The amount of peak 2 sample (Fig. 2) added was 400 lal, equal to 78 lag protein. The peak 1 sample (Fig. 2) contained 18 pg protein in 400 lal. After 1 h coupling at 20 ~ C with gentle rotation, the gel was centrifuged (10 s, 10,000 g) and the enzyme activities in the supernatant were determined.
Glucosyltransferase assays. The activity of 3 GT was determined as described earlier (Gerats et al. 1983) with delphinidin as substrate. The 5GT standard assay mixture consisted of 29 laM anthocyanin substrate dissolved in 5 mM HCt; 200 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-potassium phosphate buffer (pH 8.3); 0.8 mM UDPGIc and 45 lal enzyme extract (50-250 lag protein) in a reaction volume of 100 lal. After 2 rain of incubation at 30 ~ C, the reaction was terminated by adding 400 p.1 of a mixture containing two parts of chloroform and one part of methanol-2% HC1 (v/v). When low enzymic activities were to be expected (substrate specificity, column fractions, certain genotypes), incubations were carried out for 15-20 rain. The mixtures for determinations at different pH values were buffered with 200 mM Tris-potassium phosphate (pH 7.0-9.2) or 200raM sodimn acetate-HCl (pH 4.0-6.5). Control incubations contained no enzyme. The upper phase of the Folch partition obtained (Folch et al. J 957) was subjected to analysis using high performance liquid chromatography (HPLC). The amount of glucosylated product was quantified as described earlier Oonsson et al. ~982), using a mM extinction coefficient of 34 at 530 nm for all anthocyanins. Isolation and identification of anthocyanins. Delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-rutinoside, delphinidin 3-(p-coumaroyl)-rutinoside-5-glucoside (del 3RGac5G) and petunidin 3-(p-coumaroyl)-rutinoside-5-glucoside (pet 3RGac5G) were isolated and identified as described earlier (Jonsson et al. 1982; Schram et al. 1983). The purification of delphinidin 3-(p-coumaroyl)-rutinoside (del 3RGac) and petunidin 3-(p-coumaroyl)-rutinoside (pet 3RGac), extracted from mature pollen of various lines of Petunia, was carried out in the same way, but omitting the re-chromatography. Depending on the genetic background of the plant, the extract contained pet 3RGac or a mixture of del 3RGac and pet 3RGac (3: 1).
343
The anthocyanin extracts were analyzed by thin-layer chromatography using three different solvents, which are described in the legend to Table 1. The products after a partial acid hydrolysis of the unknown anthocyanins were identified after chromatography using solvent 3. In order to demonstrate the presence of an acyl group, the anthocyanins were treated with NaOH and thereafter chromatographed using solvent 1. Anthocyanidins were identified after acid hydrolysis of anthocyanins by chromatography on cellulose plates with acetic acid-HClwater (30:3:10, by vol.) as solvent. The HPLC analyses of anthocyanins were carried out as described previously (Jonsson et al. 1982; Schram et al. 1983). The standard 5GT assays were analyzed as follows: elution was carried out with 10% formic acid (v/v) in water with a gradient of increasing concentrations of methanol (v/v). A gradient of 22.5 to 23.7% methanol for 2.6 rain was followed by an increase to 24.5% methanol for 1.4 rain. The flow rate was 4 m l m i n -~ at 45~ and the absorbance was measured at 530 nm. For identification of the pet 3RGac derivative with the same elution program, the absorbance was measured at 540 nm and 310 nm, respectively.
Results
Identification of the 3-(p-coumaroyl)-rutinoside derivatives of anthocyanidins in pollen of Petunia hybrida. A c c o r d i n g t o t h e b i o s y n t h e t i c p a t h w a y ( F i g . 1), t h e m o s t p r o b a b l e s u b s t r a t e s f o r 5 - g l u c o sylation were either anthocyanidin 3-rutinosides or anthocyanidin 3-(p-coumaroyl)-rutinosides. The l a t t e r h a d n e v e r b e f o r e b e e n f o u n d in f l o w e r s o f Petunia hybrida, a n d n o r e f e r e n c e p i g m e n t s w e r e available from other sources either. Analyses of a n t h o c y a n i n s in c o l o u r e d m a t u r e p o l l e n f r o m v a r i o u s lines o f Petunia, h o w e v e r , r e v e a l e d t h e o c c u r rence of two unknown pigments, which were clearly separated from the main spots of the delphinidin and petunidin 3RGac5G derivatives by chromatography on silica plates with ethyl acetateformic acid-water. To identify these components they were isolated separately. After acid hydrolysis and chromatography, the anthocyanidins were i d e n t i f i e d as p e t u n i d i n a n d d e l p h i n i d i n . T h e R f values of the two unknown pigments on thin-layer chromatograms and the retention times upon H P L C a n a l y s i s a r e s h o w n i n T a b l e 1, t o g e t h e r w i t h values for some reference pigments. A further analysis o f t h e u n k n o w n p e t u n i d i n d e r i v a t i v e g a v e t h e f o l l o w i n g r e s u l t s . T h e E44 o nm/Emax, 540 n m v a l u e w a s 0.25, w h i c h i n d i c a t e d t h a t t h e r e is n o s u g a r a t p o s i t i o n 5. A p e a k a t 310 n m in t h e a b s o r p t i o n s p e c t r u m a n d t h e E31onm/E . . . . 540,m v a l u e (0.80) a r e evidence of acylation with p-coumaric acid with one molecule per anthocyanin molecule (Harborne 1967). T h e p r o d u c t a f t e r t r e a t m e n t w i t h N a O H was petunidin 3-rutinoside. After partial acid hydrolysis three components were detected: petunidin, petunidin 3-glucoside and petunidin 3-rutinoside. T h e g e n e t i c b a c k g r o u n d o f t h e a n t h e r s (An4Hfl-Rt-Gf-) f u r t h e r s u p p o r t s t h e s e r e s u l t s , since
L.M.V. Jonsson et al.: Anthocyanin 5-O-glucosyltransferase in flowers of Petunia
344
Table 1. Rf values and retention times obtained from thin-layer chromatography and HPLC analysis of the unknown delphinidin and petunidin derivatives isolated from pollen of Petunia hybrida, and comparison with known references, n.d. = not determined. Solvent 1 : ethylacetate-formic acid-water (60:12:16, by vol.) silica; solvent 2 : n-butanol-acetic acid-water (4:1 : 5, by vol.) upper phase, cellulose; solvent 3 : acetic acid-HCl-water (15 : 3 : 82, by vol.) cellulose; HPLC: standard 5 GT-program; pet = petunidin, del = delphinidin, 3RG = 3-rutinoside
Solvent 1, Rf. Solvent 2, Rf. Solvent 3, Rf. HPLC, rain
pet-x
del-x
pet3RG
del3RG
pet3RGac5G
de13RGac5G
0.74 0.70 0.39 4.60
0.70 0.67 n.d. 3.70
0.49 0.33 n.d. n.d.
0.43 0.29 0.20 1.30
0.50 n.d. n.d. 2.75
0.42 n.d. 0.41 1.75
the gene Rt controls rhamnosylation at the 3-glucose group of anthocyanins (Wiering et al. 1979). We conclude that the unknown petunidin derivative is petunidin 3RGac, and suggest that the unknown delphinidin derivative is delphinidin 3RGac.
Table 2. Substrate specificity of anthocyanin 5-O-glucosyltransferase in enzyme preparations from flower buds of Petunia hybrida, line W80
Formation of anthocyanidin 3- (p-coumaroyl)-rutinoside-5-glucoside in vitro. Crude enzyme preparations from flower buds of Petunia hybrida cata-
Petunidin 3RGac
27
29
Delphinidin 3RGac-petunidin 3RGac Delphinidin 3-rutinoside
27
29
< 0.5
20, 90
Cyanidin 3-rutinoside
< 0.5
20, 90
Delphinidin 3-glucoside
< 0.5
16
lyzed the formation in vitro of anthocyanidin 3RGac5G derivatives from anthocyanidin 3RGac and UDPGlc. The reaction was linear with time for at least 10 rain. There was also a linear relationship between the formation of product and the amount of protein in the assay to at least 1.5 mg protein m l - ~. The substrate specificity of this 5 GT activity was determined in the crude extracts and the results are shown in Table 2. The p-coumaroyl group was necessary for activity. Anthocyanins lacking the acyl-group were not accepted as substrates.
Specific activity Concen(pkat mg- z protein) tration (~M)
Substrate
Pook
:l
1
2s 20
to-
~ ->
08 -
15
o
':.
04-
Specificity of the 5GT and the 3GT. The glucosyltransferases present in crude extracts could be separated by chromatofocusing on a gradient between pH 6.2-4.0. Extracts from three different, coloured mutants (R27, M43, M73) gave similar elution profiles, one of which is shown in Fig. 2. The absence of a sharp peak of 5 GT activity in the illustrated experiment is caused by low activities hampering detection. An experiment without buffering of collected fractions allowed the direct determination of the iso-electric points of the two enzymes. These were: 5 G T : p H 4.75, 3 G T : p H 5.2. Fractions containing the two glucosyl-transferases were separately pooled as indicated in Fig. 2. The 3 GT and 5 GT preparations thus obtained had undergone a 14- and 30-fold purification, respectively, as compared with the ammonium-sulphatefractionated extract applied to the chromatofocusing column. These partially purified GT preparations were used for determination of their positionspecificities. The preparation obtained from peak 1
.....
0-
0
0.25
050
9" . .
0.75
10
Rf
Fig. 2. Elution profile of anthocyanin glucosyltransferases in flowers of Petunia hybrida, line M43, after chromatofocusing with a pH gradient between pH 6.2-4.0. (zx-zx)= 3-O-glucosyltransferase activity, (o-o)=5-O-glucosyltransferase activity, ("')=protein content, Azson~. The extract contained a t o t a l of 23.4 mg protein. Arrows indicate fractions that were pooled to obtain the partially purified GTs
was unable to glucosylate the anthocyanidin 3RGac substrate at the 5-position. There was no detectable 5-glucosyltransferase activity ( < 15 pkat mg - 1 protein), whereas the 3 GT activity was 1980 p k a t m g -1 protein. Peak2, on the other hand, showed appreciable 3GT activity (61 pkat mg -1 protein) compared with the 5GT activity: 278 pkat mg -1 protein. Apparently, this activity was low enough to have escaped detection in the original fractions before pooling.
L,M.V. Jonsson et al, : Anthocyanin 5-O-glucosyltransferase in flowers of Petunia Table 3. Effect of incubation of Peak I and Peak 2 after chromatofocusing (Fig. 2) with anti-3GT immobilized to Protein A-Sepharose CL-4B Sample
3GT-Peak
5GT-Peak
Input activity (pkat)
Input serum (mg) Normal rabbat
Anti-3GT
42.0 3GT
2.5 1.5 0.5
-
-
2.5
0
-
1.5
0
-
0.5
0
2.5 1.5 -
-2.5
-
1.5
5.4 5.2 0.2 0,4
2.5 1.5 ---
2.5 1.5
5.0 3GT
22.0 5GT
Activity in supernatant (pkat) 33.6 54.6 44.9
22.0 22.0 22.0 22.0
To investigate further whether this 3 GT activity indeed should be ascribed to the 5 GT or is the result of contamination with 3 GT, we incubated samples of the separate glucosyltransferases with antibodies raised against 3 GT (Table 3). As might be expected, the 3 GT preparation (Peak 1) had lost all activity after incubation. The 5GT activity of the Peak-2 sample, on the other hand, was not affected at all. Nevertheless, most of the 3 GT activity of the same sample was precipitated. The results imply that the Peak-2 sample contained at least two enzymes: one with 3 GT activity which was almost completely precipitated and a second enzyme with 5GT activity, which was not recognized by the antibodies. The 3GT activity of the Peak-2 sample might correspond to a third glucosyltransferase, exhibiting 3GT activity and only partially cross-reacting with the antiserum. The 3 GT activity remaining in the supernatant might also be attributed to the 5 GT enzyme. In the latter c a s e , the 3GT activity of 5GT amounts to only 0.9-1.8% of the 5GT activity.
Other properties of the anthocyanin 5-O-gIucosyltransferase. Table 4 summarizes a number of properties of 5GT, determined with the partially purfled enzyme after chromatofocusing (Fig. 2). The pH-activity optimum was found at pH 8.3, with half-maximal velocities at pH 6.5 and 9.2. After incubation for 5 rain without enzyme or without U D P G l c in the assay, more than 90% of the anthocyanin was still present, regardless of the pH at which the experiments were run. Kinetic measurements showed that the 5GT was saturated with regard to the two substrates
345
Table 4. Properties of the anthocyanin 5-O-glucosyltransferase in Petunia hybrida pH-activity optimum pl M r dalton I
Planta (1984) 160:341-347
9 Springer-Verlag 1984
Properties and genetic control of anthocyanin 5-O-glucosyltransferase in flowers of Petunia hybrida L.M.V. Jonsson, M.E.G. Aarsman, J. van Diepen, P. de Vlaming, N. Smit and A.W. Schram Research Section 'Biosynthesis of Flavonoids', Departments of Plant Physiology and Genetics, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, The Netherlands
Abstract. An anthocyanin 5-O-glucosyltransferase from flowers of Petunia hybrida was purified about 30-fold. Using uridine 5'-diphosphoglucose as glucose donor (Km 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (Km 3 gM), isolated from pollen of Petunia. Delphinidin 3-rutinoside, cyanidin 3-rutinoside and delphinidin 3-glucoside did not serve as substrates. The glucosylation of petunidin 3-(p-coumaroyl)-rutinoside showed a pH-activity optimum at pH 8.3 and was neither stimulated by Mg 2 § or Ca 2 § nor inhibited by ethylenediaminetetraacetic acid. After separating the 5-O-glucosyltransferase from the anthocyanidin 3-O-glucosyltransferase by means of chromatofocusing, it was shown that both enzymes exhibit a high degree of positional specificity. The 5-0glucosyltransferase activity was correlated with the gene Anl, but not with the gene Gf. Key words: Anthocyanin biosynthesis - Anthocyanin glucosyltransferase - Anthocyanidin 3-(/)coumaroyl)-rutinoside - Petunia.
Introduction The anthocyanin pigments, widely found in higher plants, occur in vivo as glycosides. The glycosyl groups are bound to one or more of the phenolic hydroxyl groups of the flavonoid C15-skeleton. Until now, 3-glycosides, 3,5-bisglycosides and 3,7bisglycosides have been described (Hahlbrock 1981). All anthocyanins found in flowers of Petunia hybrida are glucosylated at the 3-position. Depending on the genetic background, they may Abbreviations. HPLC=high performance liquid chromatography; 3GT = 3-O-glucosyltransferase; 5GT = 5-O-glucosyltransferase; 3RGac=3-(p-coumaroyl)-rutinoside; 3RGac5G=3-(pcoumaroyl)-rutinoside-5-glucoside; UDPGlc=uridine 5'-diphosphoglucose
also possess a glucose group at the 5-position. Other substitutions of the anthocyanin molecule involve rhamnosylation, acylation by p-coumaric acid and methylation of the B-ring. Using genetic information, the order of these modification reactions has been established (Wiering 1974). The proposed biosynthetic pathway is illustrated in Fig. 1. The glucosylation at the 3-position is carried out by an anthocyanidin 3-O-glucosyltransferase (3 GT) using uridine Y-diphosphoglucose (UDPGIc) as the glucose donor. The genetic control and some properties of this enzyme have been described (Kho et al. 1978). The level of 3GT activity is 5-20% of the normal level when one of the two genes Anl and An2 is homozygous recessive, but residual 3 GT activity was still found in plants homozygous recessive for both these genes (Gerats et al. 1983). It was suggested that an anthocyanin 5-O-glucosyltransferase (5 GT), without an absolute position specificity, might account for this residual activity. The glucosylation at the 5-position is controlled by the gene Gf (Wiering and de Vlaming 1973). Genetic evidence indicates that this pleiotropic gene controls both glycosylation at the 5-position and acylation at the 3-rhamnose group. This situation seems to exist in anthocyanin biosynthesis in Solanaceae in general; genes with effects similar to that of Gfwere found in Solanum tuberosum (Harborne 1960) as well as in Solanum melongena (Tigchelaar et al. 1968). We assume that 5-glucosylation and acylation are controlled by two different genes, and that these steps are carried out in a certain, arbitrary order. The gene Gf is supposed to control the first step, which allows the second to occur.
In order to study the specificity of the anthocyanin glucosyltransferases in Petunia hybrida and to investigate whether Gfcontrols 5-glucosylation, the anthocyanin 5GT was isolated. In this paper we report the results of biochemical studies on the properties and genetic control of the anthocyanin
L.M.V. Jonsson et al. : Anthocyanin 5-O-glucosyltransferase in flowers of Petunia
342
HO--[~O+ ~ ; H OH
grey1
O-GIc
de[phinidin 3-glucoside
.o
-ro>CG.
"OH
grey2
OH O-Gtc-O-Rha delphinidin 3-rutinoside
10f
pu,0,e O-Gtc O- 6tc-O-Rha- p.Cum delphinidin 3-(p-cumQroyl) - rutinosido-5-gtucoside
I M t l Mi2 Mf 1 Mf2
.o-rf-rO
~OCH3
o.
purp,e
~0H O-Gtc
O-Gtc-O-Rha- p.Cum
petunidin 3- (p-cumoroyl}-rutinosido - 5- glucoside
Nil Mr2 Mfl Mf2
jOCH 3 HO" - F ' ~ j ~ k / ' OH purple ~--~ "---~OCH3 O-Gtc O-Gtc-O-Rha-p.Cum molvidin 3-(p-cumoroyl}- rutinosido- 5-glucoside
Fig, 1. Pathways of glycosylation, acylation and methylation reactions in anthocyanin biosynthesis in flowers of Petunia hybrida as deduced from genetic evidence
glucosyltransferases, with special emphasis on 5GT. Materials and methods Plant material. All plants (Petunia hybrida hort.) were grown under greenhouse conditions. The genotypes of the different inbred lines are given in Table 5.
Enzyme extraction. All steps were performed at 0-4 ~ C. Limbs of flowers and flower buds were homogenized with 100 mM potassium phosphate, pH 8.5, containing ~.4 mM fl-mercaptoethanol (5 ml buffer per 25 limbs) and some quartz sand in a mortar. During homogenization, Dowex 1X2-200 (Sigma, St. Louis, Mo., USA) at half the amount of the fresh weight of the tissue was added. For the study of 5GT activity in different mutants, only buds of 15-35 mm length (measured from the base of the corolla) were used. The homogenate was centrifuged at 38,000 g for 20 min. An aliquot of the supernatant liquid (2.5 ml) was chromatographed on a Sephadex G-25 column (1.5 cm diameter, 8.3 cm long; Pharmacia, Uppsala, Sweden), which had been pre-equilibrated with 100 mM potassium phosphate, pH 7.7. The same buffer was used for elution, and fractions containing protein were pooled for assay of 5GT activity and protein content.
Protein was determined according to Bradford (1976) with bovine serum albumin as reference protein.
Molecular weight determination. The molecular weights of glucosyltransferases were determined with crude enzyme preparations after Sephadex G-25 chromatography, using a Sephacryl S-200 Superfine (Pharmacia) column (1.6 cm diameter, 60 cm long) with 100 mM potassium phosphate, pH 7.7, as elution buffer. The column was calibrated with lysozyme (M~ 14,300), trypsinogen (M r 24,000), chymotrypsinogen (M r 25,000), ovalbumin (M r 45,000), bovine serum albumin (M, 66,000) and aldolase (M, 158,000). Chromatofocusing of enzymes. Proteins were precipitated from supernatant liquid (see preceeding section) by ammonimn sulphate at ~ 4 ~ C. The supernatant was brought to 80% saturation by adding solid (NH4)2SO4 over a period of 20 min, during which the pH of the solution was maintained at pH 7.3-7.6 by adding drops of 1 M KOH. The suspension was centrifuged at 38,000 g for 20 min, and the resulting pellet was resuspended in 25 mM histidine-HC1 pH 6.2. After recentrifugation under identical conditions, the final supernatant liquid was applied to a Sephadex G-25 column (pre-equilibrated and eluted with 25 mM histidine-HC1, pH 6.2) to remove residual (NH4)2SO 4. The protein-containing fractions were then applied to a PBE 94 (Pharmacia) chromatofocusing column (0.55cm diameter, 17 cm long), which had been pre-equilibrated with 25 mM histidine-HC1, pH 6.2. The column was eluted with Polybuffer 74 (Pharmacia), pH 4.0, at a flow rate of 35 ml h-1. Fractions of 2.0 ml were collected in tubes containing 100 ~tl 2 M potassium phosphate, pH 8.1. Pooled fractions were concentrated using an ultrafiltration cell (Amicon, Lexington, USA), provided with a PM-10 membrane~ The enzyme preparations used for determining the pH-activity profile were applied to a Sephadex G-25 column and eluted with 10 mM potassium phosphate, pH 7.7. Partial purification of 3-O-glucosyltransferase. The white-flowering mutants W37 and W39 were used as source of enzyme. These lines are homozygous recessive for the gene An3 and blocked in an early step of anthocyanin biosynthesis (Wiering et al. 1979) but contain normal levels of 3GT-activity (Kho et al. 1978). All steps were performed at 0-4 ~ C. For one isolation 25 g (fresh weight) limbs of flowers and flower buds were homogenized in a mortar with some sand and 60 ml 10 mM potassium phosphate buffer pH 7.5, containing 20 m M fl-mercaptoethanol and 5% polyvinylpyrrolidone (w/v). The homogenate was centrifuged at 38,000 g during 20 min. A second extraction of the pellet using 30 ml buffer and re-centrifugation (38,000 g, 20 min) followed. The supernatants were combined and applied to a polyvinylpolypyrrolidone column (12.0 cm long, 5.8 cm diameter), pre-equilibrated and eluted with 10 mM potassium phosphate (pH 7.5). Protein-containing fractions were pooled and dialyzed during 16 h against 10 mM potassium phosphate buffer pH 7.0 and subsequently applied to a DEAEcellulose column (2.4 cm diameter, 6.5 cm long), pre-equilibrated with the same buffer. The bound protein was eluted with 300 ml of a linear gradient of 0 to 1.0 M NaC1 in 10 mM potassium phosphate (pH 7.0). Fractions containing 3GT-activity were pooled and dialyzed against 25 mM histidine-HC1 pH 6.2 and thereafter subjected to chromatofocusing on a PBE 94 column (Pharmacia) as described in the preceeding section. The fractions containing 3GT-activity were pooled and concentrated using an ultrafiltration cell (Amicon), provided with a UM-2 membrane. The enzyme was dialyzed against water and thereafter lyophilized.
Preparation of antiserum. 3 GT was purified as described above. 300 I~g lyophilized enzyme protein dissolved in 1 ml phosphatebuffered saline (pH 7.4) was mixed with 1 ml complete Freunds adjuvant and injected intramuscularly in a rabbit at four differ-
L.M.V. Jonsson et al. : Anthocyanin 5-O-glucosyltransferase in flowers of Petunia ent sites. Three booster injections with the same enzyme preparation, mixed with 1 ml incomplete Freunds adjuvant were given three, five and seven weeks later. In the l l t h week a booster of 600 lag protein was given and in the 12th week the rabbit was plasmaphoresed. A new injection of 250 lag protein, mixed with 1 ml incomplete Freunds adjuvant was given in the 20th week. After two more weeks the rabbit was again plasmaphoresed (50 ml). Complement was inactivated by incubation of the antiserum at 56~ C for 30 min. A crude immunoglobulin fraction was obtained by precipitation with (NH4)2SO 4 to 50% saturation, followed by dialysis against phosphate-buffered saline (pH 7.4).
Incubation of enzyme with antiserum immobilized to Protein ASepharose CL-4B. The coupling of anti-3 GT to Protein A-Sepharose CL-4B (Pharmacia) was carried out at 20 ~ C for 2 h, with gentle rotation in Eppendorf tubes. To each tube containing 7.5 mg Protein A-Sepharose CL-4B was added 0.5, 1.5 or 2.5 mg anti-3GT serum-protein in 250 lal phosphate-buffered saline (pH 7.4). Control incubations contained corresponding amounts of normal rabbit serum. The gel was washed three times with phosphate-buffered saline (pH 7.4) by mixing on a vortex mixer and centrifugation (10 s, 10,000 g). To prevent inhibition of immunoprecipitation caused by Polybuffer, the enzyme samples were applied to a Sephadex G-25 column and eluted with 100 mM potassium phosphate (pH 7.7) before incubation with immobilized antiserum. The amount of peak 2 sample (Fig. 2) added was 400 lal, equal to 78 lag protein. The peak 1 sample (Fig. 2) contained 18 pg protein in 400 lal. After 1 h coupling at 20 ~ C with gentle rotation, the gel was centrifuged (10 s, 10,000 g) and the enzyme activities in the supernatant were determined.
Glucosyltransferase assays. The activity of 3 GT was determined as described earlier (Gerats et al. 1983) with delphinidin as substrate. The 5GT standard assay mixture consisted of 29 laM anthocyanin substrate dissolved in 5 mM HCt; 200 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-potassium phosphate buffer (pH 8.3); 0.8 mM UDPGIc and 45 lal enzyme extract (50-250 lag protein) in a reaction volume of 100 lal. After 2 rain of incubation at 30 ~ C, the reaction was terminated by adding 400 p.1 of a mixture containing two parts of chloroform and one part of methanol-2% HC1 (v/v). When low enzymic activities were to be expected (substrate specificity, column fractions, certain genotypes), incubations were carried out for 15-20 rain. The mixtures for determinations at different pH values were buffered with 200 mM Tris-potassium phosphate (pH 7.0-9.2) or 200raM sodimn acetate-HCl (pH 4.0-6.5). Control incubations contained no enzyme. The upper phase of the Folch partition obtained (Folch et al. J 957) was subjected to analysis using high performance liquid chromatography (HPLC). The amount of glucosylated product was quantified as described earlier Oonsson et al. ~982), using a mM extinction coefficient of 34 at 530 nm for all anthocyanins. Isolation and identification of anthocyanins. Delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-rutinoside, delphinidin 3-(p-coumaroyl)-rutinoside-5-glucoside (del 3RGac5G) and petunidin 3-(p-coumaroyl)-rutinoside-5-glucoside (pet 3RGac5G) were isolated and identified as described earlier (Jonsson et al. 1982; Schram et al. 1983). The purification of delphinidin 3-(p-coumaroyl)-rutinoside (del 3RGac) and petunidin 3-(p-coumaroyl)-rutinoside (pet 3RGac), extracted from mature pollen of various lines of Petunia, was carried out in the same way, but omitting the re-chromatography. Depending on the genetic background of the plant, the extract contained pet 3RGac or a mixture of del 3RGac and pet 3RGac (3: 1).
343
The anthocyanin extracts were analyzed by thin-layer chromatography using three different solvents, which are described in the legend to Table 1. The products after a partial acid hydrolysis of the unknown anthocyanins were identified after chromatography using solvent 3. In order to demonstrate the presence of an acyl group, the anthocyanins were treated with NaOH and thereafter chromatographed using solvent 1. Anthocyanidins were identified after acid hydrolysis of anthocyanins by chromatography on cellulose plates with acetic acid-HClwater (30:3:10, by vol.) as solvent. The HPLC analyses of anthocyanins were carried out as described previously (Jonsson et al. 1982; Schram et al. 1983). The standard 5GT assays were analyzed as follows: elution was carried out with 10% formic acid (v/v) in water with a gradient of increasing concentrations of methanol (v/v). A gradient of 22.5 to 23.7% methanol for 2.6 rain was followed by an increase to 24.5% methanol for 1.4 rain. The flow rate was 4 m l m i n -~ at 45~ and the absorbance was measured at 530 nm. For identification of the pet 3RGac derivative with the same elution program, the absorbance was measured at 540 nm and 310 nm, respectively.
Results
Identification of the 3-(p-coumaroyl)-rutinoside derivatives of anthocyanidins in pollen of Petunia hybrida. A c c o r d i n g t o t h e b i o s y n t h e t i c p a t h w a y ( F i g . 1), t h e m o s t p r o b a b l e s u b s t r a t e s f o r 5 - g l u c o sylation were either anthocyanidin 3-rutinosides or anthocyanidin 3-(p-coumaroyl)-rutinosides. The l a t t e r h a d n e v e r b e f o r e b e e n f o u n d in f l o w e r s o f Petunia hybrida, a n d n o r e f e r e n c e p i g m e n t s w e r e available from other sources either. Analyses of a n t h o c y a n i n s in c o l o u r e d m a t u r e p o l l e n f r o m v a r i o u s lines o f Petunia, h o w e v e r , r e v e a l e d t h e o c c u r rence of two unknown pigments, which were clearly separated from the main spots of the delphinidin and petunidin 3RGac5G derivatives by chromatography on silica plates with ethyl acetateformic acid-water. To identify these components they were isolated separately. After acid hydrolysis and chromatography, the anthocyanidins were i d e n t i f i e d as p e t u n i d i n a n d d e l p h i n i d i n . T h e R f values of the two unknown pigments on thin-layer chromatograms and the retention times upon H P L C a n a l y s i s a r e s h o w n i n T a b l e 1, t o g e t h e r w i t h values for some reference pigments. A further analysis o f t h e u n k n o w n p e t u n i d i n d e r i v a t i v e g a v e t h e f o l l o w i n g r e s u l t s . T h e E44 o nm/Emax, 540 n m v a l u e w a s 0.25, w h i c h i n d i c a t e d t h a t t h e r e is n o s u g a r a t p o s i t i o n 5. A p e a k a t 310 n m in t h e a b s o r p t i o n s p e c t r u m a n d t h e E31onm/E . . . . 540,m v a l u e (0.80) a r e evidence of acylation with p-coumaric acid with one molecule per anthocyanin molecule (Harborne 1967). T h e p r o d u c t a f t e r t r e a t m e n t w i t h N a O H was petunidin 3-rutinoside. After partial acid hydrolysis three components were detected: petunidin, petunidin 3-glucoside and petunidin 3-rutinoside. T h e g e n e t i c b a c k g r o u n d o f t h e a n t h e r s (An4Hfl-Rt-Gf-) f u r t h e r s u p p o r t s t h e s e r e s u l t s , since
L.M.V. Jonsson et al.: Anthocyanin 5-O-glucosyltransferase in flowers of Petunia
344
Table 1. Rf values and retention times obtained from thin-layer chromatography and HPLC analysis of the unknown delphinidin and petunidin derivatives isolated from pollen of Petunia hybrida, and comparison with known references, n.d. = not determined. Solvent 1 : ethylacetate-formic acid-water (60:12:16, by vol.) silica; solvent 2 : n-butanol-acetic acid-water (4:1 : 5, by vol.) upper phase, cellulose; solvent 3 : acetic acid-HCl-water (15 : 3 : 82, by vol.) cellulose; HPLC: standard 5 GT-program; pet = petunidin, del = delphinidin, 3RG = 3-rutinoside
Solvent 1, Rf. Solvent 2, Rf. Solvent 3, Rf. HPLC, rain
pet-x
del-x
pet3RG
del3RG
pet3RGac5G
de13RGac5G
0.74 0.70 0.39 4.60
0.70 0.67 n.d. 3.70
0.49 0.33 n.d. n.d.
0.43 0.29 0.20 1.30
0.50 n.d. n.d. 2.75
0.42 n.d. 0.41 1.75
the gene Rt controls rhamnosylation at the 3-glucose group of anthocyanins (Wiering et al. 1979). We conclude that the unknown petunidin derivative is petunidin 3RGac, and suggest that the unknown delphinidin derivative is delphinidin 3RGac.
Table 2. Substrate specificity of anthocyanin 5-O-glucosyltransferase in enzyme preparations from flower buds of Petunia hybrida, line W80
Formation of anthocyanidin 3- (p-coumaroyl)-rutinoside-5-glucoside in vitro. Crude enzyme preparations from flower buds of Petunia hybrida cata-
Petunidin 3RGac
27
29
Delphinidin 3RGac-petunidin 3RGac Delphinidin 3-rutinoside
27
29
< 0.5
20, 90
Cyanidin 3-rutinoside
< 0.5
20, 90
Delphinidin 3-glucoside
< 0.5
16
lyzed the formation in vitro of anthocyanidin 3RGac5G derivatives from anthocyanidin 3RGac and UDPGlc. The reaction was linear with time for at least 10 rain. There was also a linear relationship between the formation of product and the amount of protein in the assay to at least 1.5 mg protein m l - ~. The substrate specificity of this 5 GT activity was determined in the crude extracts and the results are shown in Table 2. The p-coumaroyl group was necessary for activity. Anthocyanins lacking the acyl-group were not accepted as substrates.
Specific activity Concen(pkat mg- z protein) tration (~M)
Substrate
Pook
:l
1
2s 20
to-
~ ->
08 -
15
o
':.
04-
Specificity of the 5GT and the 3GT. The glucosyltransferases present in crude extracts could be separated by chromatofocusing on a gradient between pH 6.2-4.0. Extracts from three different, coloured mutants (R27, M43, M73) gave similar elution profiles, one of which is shown in Fig. 2. The absence of a sharp peak of 5 GT activity in the illustrated experiment is caused by low activities hampering detection. An experiment without buffering of collected fractions allowed the direct determination of the iso-electric points of the two enzymes. These were: 5 G T : p H 4.75, 3 G T : p H 5.2. Fractions containing the two glucosyl-transferases were separately pooled as indicated in Fig. 2. The 3 GT and 5 GT preparations thus obtained had undergone a 14- and 30-fold purification, respectively, as compared with the ammonium-sulphatefractionated extract applied to the chromatofocusing column. These partially purified GT preparations were used for determination of their positionspecificities. The preparation obtained from peak 1
.....
0-
0
0.25
050
9" . .
0.75
10
Rf
Fig. 2. Elution profile of anthocyanin glucosyltransferases in flowers of Petunia hybrida, line M43, after chromatofocusing with a pH gradient between pH 6.2-4.0. (zx-zx)= 3-O-glucosyltransferase activity, (o-o)=5-O-glucosyltransferase activity, ("')=protein content, Azson~. The extract contained a t o t a l of 23.4 mg protein. Arrows indicate fractions that were pooled to obtain the partially purified GTs
was unable to glucosylate the anthocyanidin 3RGac substrate at the 5-position. There was no detectable 5-glucosyltransferase activity ( < 15 pkat mg - 1 protein), whereas the 3 GT activity was 1980 p k a t m g -1 protein. Peak2, on the other hand, showed appreciable 3GT activity (61 pkat mg -1 protein) compared with the 5GT activity: 278 pkat mg -1 protein. Apparently, this activity was low enough to have escaped detection in the original fractions before pooling.
L,M.V. Jonsson et al, : Anthocyanin 5-O-glucosyltransferase in flowers of Petunia Table 3. Effect of incubation of Peak I and Peak 2 after chromatofocusing (Fig. 2) with anti-3GT immobilized to Protein A-Sepharose CL-4B Sample
3GT-Peak
5GT-Peak
Input activity (pkat)
Input serum (mg) Normal rabbat
Anti-3GT
42.0 3GT
2.5 1.5 0.5
-
-
2.5
0
-
1.5
0
-
0.5
0
2.5 1.5 -
-2.5
-
1.5
5.4 5.2 0.2 0,4
2.5 1.5 ---
2.5 1.5
5.0 3GT
22.0 5GT
Activity in supernatant (pkat) 33.6 54.6 44.9
22.0 22.0 22.0 22.0
To investigate further whether this 3 GT activity indeed should be ascribed to the 5 GT or is the result of contamination with 3 GT, we incubated samples of the separate glucosyltransferases with antibodies raised against 3 GT (Table 3). As might be expected, the 3 GT preparation (Peak 1) had lost all activity after incubation. The 5GT activity of the Peak-2 sample, on the other hand, was not affected at all. Nevertheless, most of the 3 GT activity of the same sample was precipitated. The results imply that the Peak-2 sample contained at least two enzymes: one with 3 GT activity which was almost completely precipitated and a second enzyme with 5GT activity, which was not recognized by the antibodies. The 3GT activity of the Peak-2 sample might correspond to a third glucosyltransferase, exhibiting 3GT activity and only partially cross-reacting with the antiserum. The 3 GT activity remaining in the supernatant might also be attributed to the 5 GT enzyme. In the latter c a s e , the 3GT activity of 5GT amounts to only 0.9-1.8% of the 5GT activity.
Other properties of the anthocyanin 5-O-gIucosyltransferase. Table 4 summarizes a number of properties of 5GT, determined with the partially purfled enzyme after chromatofocusing (Fig. 2). The pH-activity optimum was found at pH 8.3, with half-maximal velocities at pH 6.5 and 9.2. After incubation for 5 rain without enzyme or without U D P G l c in the assay, more than 90% of the anthocyanin was still present, regardless of the pH at which the experiments were run. Kinetic measurements showed that the 5GT was saturated with regard to the two substrates
345
Table 4. Properties of the anthocyanin 5-O-glucosyltransferase in Petunia hybrida pH-activity optimum pl M r dalton I
