Isolation and characterization of a UDP-glucose: cyanidin 3-O-glucosyltransferase from grape cell suspension cultures (Vitis vinifera L.)

plANk- .

)CIENCE

Plant Science 112 (1995) 43-51

Isolation and characterization of a UDP-glucose:cyanidin 3-0-glucosyltransferase from grape cell suspension cultures (Vilis vinifera L.) Chi Bao Do, Fran90is Cormier*, Yves Nicolas 1 Food Research and Development Centre. Agriculture Agri-Food Canada. 3600 Casavant Blvd. West. St-Hyacinthe. Quebec. Canada J2S 8E3 Received 28 February 1995; revision received 28 July 1995; accepted 12 September 1995

Abstract An enzyme catalyzing the transfer of the g1ucosyl moiety of UDP-g1ucose to the 3-hydroxyl group of cyanidin has been isolated from a Vitis vinifera cell suspension culture. The enzyme was purified 75-fold by ion-exchange, chromatofocusing and gel filtration liquid chromatography with a recovery of 3.8%. The enzyme had a pH optimum at 8.0. The g1ucosyltransferase showed the highest activity with cyanidin as acceptor but also utilized delphinidin to a significant degree. Pelargonidin, peonidin and malvidin were glucosylated but at a lower rate. An apparent Km value of 1.2 mM for UDP-g1ucose was determined. At an equal concentration of UDP-glucose the apparent Km values of the enzyme for the acceptors were 18 ILM for cyanidin (3', 4' OH) and 28 ILM for delphinidin (3', 4', 5' OH). The purified enzyme had an isoelectric point of 4.55 and a molecular weight of 56 kDa. Substrate affinity of the enzyme indicates that it is the first step of two anthocyanin branches which begin with cyanidin 3-glucoside and delphinidin 3-g1ucoside. Also, the lower activity of the enzyme with 3' - and 5' -O-methylated derivatives of cyanidin and delphinidin would explain in part why methylation occurs after the glucosylation step. Keywords: UDP-glucose:cyanidin 3-0-glucosyltransferase; Glucosyltransferase; Purification; Anthocyanin biosynthesis; Vitis vinifera; Cell cultures

l. Introduction

The anthocyanidin 3-0-monoglucosides are the main products accumulated by Vilis vinifera cell cultures [1-3J. In most plant sources, glycosyla• Corresponding author, Fax: 514 773 8461; Internet: [email protected]. I Present address: Laboratoire de Biochimie et Technologie des Proteines, I.N.R.A., Rue de la Geraudiere, 44026 Nantes CEDEX 03, France.

tion, usually a glucosylation, in the 3-position of anthocyanidin is an obligatory reaction leading to the formation of the first stable anthocyanin [4J. Because of the instability of the aglycones, lack of 3-0-glycosyltransferase would be expected to prevent accumulation of anthocyanins and it is assumed that the anthocyanidin formed is immediately glycosylated at the 3-hydroxyl group [5J. These glycosides are substrates for further glycosylation, methylation and acylation of the sugar

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C.B. Do el al. / Plant Science 112 (1995) 43-51

residues [6]. Glycosyltransferases have been isolated and characterized from many plant sources such as seedlings [7-11], cell suspensions [12-14], flower part [15,16], leaves [17], roots [18] and fruit [19]. An enzyme catalyzing the glucosylation of the 3-hydroxyl group of cyanidin was first demonstrated in red cabbage seedlings [8] and in Haplopappus gracilis [12]. This enzyme preparation was not specific for cyanidin, but glucosylated a variety of flavonoid compounds containing a 3hydroxyl group. The enzyme showed similar properties with glucosyltransferases from other plants that exhibited a broad substrate specificity. The enzyme was also isolated from Silene dioica [20,21] and Petunia hybrida [22]. Extensive studies on different genotypes of Petunia hybrida [23] and Mathiola incana [6] have shown that the 3-0glucosyltransferases for flavonoids and anthocyanidins are identical. However, the flavonol 3O-glucosyltransferase from tulip anther does not glucosylate anthocyanidins, but is highly specific for flavonols [15]. To better understand the role and specificity of the glucosyltransferases in the metabolism of anthocyanins, these enzymes were investigated in Vitis vinifera cell suspensions. In this paper, the purification of a Vilis vinifera glucosyltransferase is described and some of its major kinetic and molecular properties are characterized.

2. Materials and methods 2.1. Cell cultures Experiments were performed using Vitis vinifera cv. Gamay Freaux cell suspension line no. 15.1. Cell suspension culture was grown in B-5 medium [24] as described previously [25]. Seven-day-old cells were harvested by filtration. Protein extraction from the cells was carried out as described below. 2.2. Chemicals, reagents and equipment For purification of the enzyme, a Pharmacia Biopilot column (Q Sepharose 351100, 35 mm x 100 mm) connected to a preparative HPLC (System Gold, Beckman, California, USA) was used for ion-exchange chromatography. A column Mono P (HR 5/20, 5 mm x 200 mm) connected to

a fast protein liquid chromatography system (Pharmacia, Uppsala, Sweden) was used for chromatofocusing. A column Shodex Pak (KW803 8/300, 8 mm x 300 mm, Millipore, USA) connected to an analytical HPLC (System Gold, Beckman) was used for gel filtration chromatography. Gel electrophoresis was performed on a Phast System (Pharmacia) using SDS (12.5%) and isoelectric focusing (PH 4-6.5) mini-gels and on a Multiphor II System (LKB) using Ampholine PAGplate (Pharmacia) 1 mm-thick gels (pH 4.0-6.5). Gels, staining dye (silver nitrate), molecular weight and isoelectric markers for the electrophoresis were supplied by Pharmacia (Montreal, PQ, Canada). Anthocyanins and anthocyanidins were from Extrasynthese (Genay, France). UDP-glucose was from Boehringer (Germany). All other chemicals were of analytical reagent grade.

2.3. Extraction and purification of enzyme Buffers. All buffers contained 10 mM polyethylene glycol 3400, 20 mM Na-diethyldithiocarbamate, 2 mM dithiothreitol and 14 mM {jmercaptoethanol unless stated otherwise. The following buffers were used: A, 100 mM Tris-HCI (pH 8.0); B, 250 mM Tris-HCI (PH 7.5); C, 25 mM Tris-HCI (PH 7.5); D, 20 mM Tris-HCl (PH 7.5); E, 50 mM Na-phosphate (PH 6.2); F, 25 mM Bis.Tris-HCI (pH 6.3); G, 100 mM Tris-HCl (pH 7.5). Glucosyltransferase assays. Different assays were carried out to determine the optimal conditions for enzyme activity. The standard reaction mixture consisted of, in a final volume of 200 1'1, buffer A, 0.086 mM cyanidin chloride, 9 mM UDP-glucose and 50 1'1 enzyme extract (20-30 I'g protein). The reaction was initiated by adding enzyme extract. The mixtures were incubated for 3 and 15 min at 30°C. The reaction was terminated by adding 100 1'1 HCl (5%). Samples were run in duplicate. Peaks were integrated for quantitative determination using cyanidin 3-g1ucoside as calibration standard. Enzyme activity was defined as moles of cyanidin 3-glucoside produced per s (katal) under the assay conditions. The product of the glucosylation of cyanidin was identified as cyanidin 3-0-glucoside by co-chromatography

C.B. Do et 01. / P/(w Science 112 (1995) 43-51

with an authentic sample using a HPLC procedure described previously [27J. Extraction. Fresh cells were washed with water, filtered, frozen in liquid nitrogen and lyophilized. All procedures for extraction and purification were carried out at 4°C or on ice. For enzyme extraction 50 g of lyophilized cells were ground with 100 g of insoluble polyvinyl pyrrolidone in 2 I of extracting solution buffer B. After 30 min of incubation under continuous stirring, the extract was separated from the residue by centrifugation at 37 800 x g for 45 min. Ammonium sulfate was added to the supernatant with gentle stirring. The fraction precipitating at 30% saturation was eliminated by centrifugation for 20 min at 6000 x g. The resulting supernatant was passed through POlO columns (Sephadex G-25, Pharmacia) equilibrated beforehand with buffer C. Glycerol was added to the eluent to a final concentration of 10%. This constituted the desalted crude extract for enzyme purification. Ion-exchange chromatography. The ion-exchange column was equilibrated with buffer D. Portions (8 mI) of desalted crude extract containing 45-50 mg of protein were injected onto the column. The enzyme was eluted with a linear gradient of NaCI (0.2 to 1 M) at a flow rate of 3 mVmin. Collected fractions of eluent (3 ml) were immediately desalted using POlO columns equilibrated with buffer E and glucosyltransferase activity was tested in each fraction. Fractions of several chromatographic runs were pooled, concentrated by ultrafiltration using Centriprep membrane (exclusion limit 30 kDa, Amicon Corp., Toronto, Canada) and assayed for protein and enzyme activity. The concentrated enzyme was further subjected to a second ion-exchange chromatography procedure which used buffer E for column equilibration. The enzyme was eluted with a linear gradient of NaCI (0.4 to I M) at a flow rate of 3 mVmin. Fractions which contained enzyme activity were pooled, concentrated and equilibrated in buffer F. Chromatofocusing. The enzyme was applied to the chromatofocusing column after equilibration with buffer F. The proteins were eluted with a linear pH gradient of 6.3 to 4.0 at a flow rate of 0.4 mVmin. The pH gradient was generated with a

45

mixture of ampholyte polybuffer 7 to 4 (Pharmacia) previously adjusted to pH 4.0 with HCI (6 M). The pH of the eluting fractions was adjusted immediately to pH 7.0 by the addition of Tris-HCI buffer to a final concentration of 100 mM. Polybuffer was removed from the active fractions by passage through POlO columns after equilibration with buffer G. The enzyme was concentrated by ultrafiltration using a Centriprep membrane. Gel filtration. Molecular sieve chromatography was performed with a Shodex Protein column (exclusion limit 150 kOa). Portions (100 "I) of concentrated enzyme were injected onto the column and eluted with Tris-HCI (100 mM, pH 7.5) buffer at a flow rate of 0.5 mVmin. Active fractions of several runs were pooled, concentrated and assayed for protein and enzyme activity. Protein assays. Protein content of the preparations was estimated using a protein assay kit (BioRad, California, USA) with 'Y-globulin as the calibration standard [26J.

2.4. Properties of partly purified enzyme Partially purified enzyme extracts from ionexchange and chromatofocusing chromatography were used to determine (I) the effects of pH, (2) the enzyme kinetics and (3) the substrate specificity. Enzyme purified by gel filtration was used to determine molecular weight and isoelectric point. Effect of pH. The effect of pH on enzyme activity was determined in 100 mM citric acid-Nar phosphate (PH 5.8-6.6), 100 mM Na-phosphate (pH 7.0-7.6), 100 mM Tris-HCI (pH 8.0-8.4) and 100 mM glycine-NaOH (pH 9.0-10.6) buffers at 30°C. The appropriate buffer containing 10 mM polyethylene glycol 3400, 20 mM Na-diethyldithiocarbamate, 2 mM dithiothreitol and 14 mM 13mercaptoethanol was used and enzyme activity was assayed under standard conditions. Substrate specificity. Specificity of the glucosyltransferase was studied using various substrates (cyanidin, delphinidin, pelargonidin, malvidin, kaempferol and quercetin) and enzyme activity was assayed under standard conditions. The product of the reaction, anthocyanidin 3-glucoside, was identified by its retention time using HPLC as described [27J. Authentic samples were available

46

C.B. Do el al. / Plant Science 112 (1995) 43-51

for cyanidin, pelargonidin, peonidin, malvidin, kaempferol and quercetin 3-glucosides. Enzyme activities were expressed as a percentage of the reaction rate for cyanidin 3-glucoside. Enzyme kinetics. The enzyme solution was incubated with various concentrations of substrates yielding a range of final concentrations from 2-70 I'M for cyanidin, 3-80 I'M for delphinidin and 0.5-15 mM for UDP-glucose. The products of the reactions were identified by reversed phase HPLe using authentic standards [27]. Integrated peaks of cyanidin 3-glucoside and delphinidin 3-glucoside were used for quantitative determination. Lineweaver-Burk plots were constructed and the Michaelis-Menten constant (KnJ and maximal velocity (Vmax) were computed. For best precision in determining the Km value, only initial enzyme velocity values at substrate concentration ranging from 20-80% saturation were used. Estimation of molecular weight. Molecular weight was determined on 12.5% SDS-PAGE. Mini-gels (5 cm x 4 cm) were run on Phast Electrophoresis System (Pharmacia) from 30-35 min at 15°e and 30 Vfcm. Low molecular weight proteins: a-lactalbumin (14.4 kDa), soybean trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and phosphorylase b (94 kDa) were used to estimate the molecular mass of the purified enzyme. Isoelectric point. A rough estimation of the isoelectric point was made by separating the proteins of a crude preparation on an Ampholine PAGplate in the pH range of 4.0-6.5 (Pharmacia) at 1200 V, 20 rnA and 8 W at 3°e for 1 h. Follow-

ing isoelectric focusing, the pH was measured along the gel using a surface electrode (Ingold) and the gel was cut into portions corresponding to - 0.2 pH units. For each portion, the gel was separated from the plastic backing and incubated in 600 1'1 of cold buffer B for 2 h. The glucosyltransferase activity of the supernatant was determined as described above. Isoelectric focusing of the purified enzyme was carried out on a 5% acrylamide mini-gel (Pharmacia) at 2000 V, 5 rnA, 3.5 W at 15°e for 30-35 min using the Phast System. Pharmalytes in the pH range of 4.0-6.5 were used as carrier ampholytes. Isoelectric point (PI) of the purified enzyme was estimated with the following reference proteins: glucose oxidase (PI 4.15), soybean trypsin inhibitor (PI 4.55), ~-galactoglobulin A (pI 5.20), bovine carbonic anhydrase B (pI 5.85) and human carbonic anhydrase B (PI 6.55). 3. Results

3.1. Enzyme purification The results presented here show for the first time the characteristics of partially purified UDPglucose:cyanidin 3-0-glucosyltransferase from a suspension of Vitis vinifera. The purification procedure, involving (NH4hS04 fractionation, chromatography on Sepharose, chromatofocusing and gel filtration resulted in a 75-fold enrichment of specific glucosyltransferase activity (Table 1). At 30% ammonium sulfate saturation, almost all of the enzyme activity could be recovered in the supernatant with 20% removal of crude extract protein. This step also offered a convenient way to

Table I Purification of UDP-g1ucose:cyanidin 3-0-g1ucosyltransferase isolated from Vilis vinifera cell cultures Purification steps

Total protein (mg)

Specific activity (nkatal/mg protein)

Crude extract (NH.v~04 + PDIO Anion exchange I Anion exchange II Chromatofocusing Gel filtration

270 219 51 32

2.5 3.0 7.2 10 28 187

1.3

0.13

Purification (-fold)

I

1.2 2.9 4.0 II

75

Recovery (%)

100 98 53 47 5.5 3.8

47

C.B. Do et al. / Plant Science 112 (1995) 43-51 14

enzyme activity during chromatography on the strong anion-exchange column and subsequent concentration. At the chromatofocusing step, the enzyme peak was eluted at around pH 4.5. All purification steps to this point resulted in an - II-fold purification of the enzyme. Further purification of the glucosyltransferase was achieved by gel filtration on a protein KW-803 column. Only one fraction showed enzyme activity. However, SDSPAGE indicated that the enzyme preparation was not homogeneous. At this point, the enzyme was purified -75-fold.

•

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8

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6

U ., Q)

E

~ c

4

UJ

3.2. Properties of the glucosyltransferase Effect of pH. Effect of pH on glucosyltransfer-

2

0 5

6

7

8

9

10

11

pH

Fig. I. pH optimum of UDP-g1ucose:cyanidin 3-0-g1ucosyltransferase isolated from Vitis vinifera cell cultures.

CYANIDIN

remove phenolic and anthocyanin compounds which, at high concentration in Vilis vinifera cell cultures, combined to proteins of the crude extract. The two-step ion-exchange chromatography at the initial stages of purification resulted in 90% removal of crude extract protein. The enzyme eluted from this column in the 0.6-0.7 M NaCI concentration ranges. However, specific activity of the preparation increased only by about 4-fold. This non-proportional increase indicated loss of

DELPHINIDIN

Table 2 Substrate specificity of cyanidin 3-0-g1ucosyltransferase from Vitis vinifera cell culture Substrate"

Cyanidin Delphinidin Pelargonidin Peonidin Malvidin Kaempferol Quercetin

Specific activity (nkataI/mg protein)

17 16 12 9.0 1.7 0 0

"Chemical structures are shown in Fig. 2.

PELARGONIDIN

~

vP

PEONIDIN

MALVIDIN

HO

3

Relative activity (% of control)

KAEMPFEROL 100 95 72

52 \0

0 0

QUERCETIN

#011 01

HO

Cf.

Fig. 2. Chemical structures of enzyme substrates listed in Table 2.

48

C.B. Do et al. / Plant Science 112 (1995) 43-51

ase activity was examined at pH values ranging from 5.8-10.6. Maximal glucosylating activity was achieved at pH 8.0 (Fig. 1). Half-maximal activity was determined at pHs 7.0 and 9.4. Substrate specificity. Various aglycones were employed for evaluation of substrate specificity of the glucosyltransferase. The glucosylation by the enzyme of various anthocyanidins led to the formation of the respective 3-glucoside and is summarized in Table 2 and Fig. 2. In general, the enzyme glucosylated only anthocyanidins and no activity was detected with flavonols (kaempferol and quercetin). The best acceptor was cyanidin. The enzyme also showed a very high activity with delphinidin and could glucosylate pelargonidin, peonidin and malvidin at a lower rate. Enzyme kinetics. The apparent Km and Vmax of cyanidin 3-0-glucosyltransferase, as determined for cyanidin at pH 8.0 and 30°C, were calculated as 18 ,...M and 21 nkatlmg protein, respectively (Fig. 3). The production of cyanidin 3-0-glucoside increased between 3 and 50 ,...M cyanidin. Under such conditions, the Km for UDP-glucose was 1.2 mM with cyanidin as an acceptor as determined from Lineweaver-Burk plot, which was linear from 0.5 to 7 mM (Fig. 4). The kinetics of delphinidin

18,-----------------.--------------~

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~ ~

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J

20

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i

9

CII

E

~

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6

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·8

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~ w

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12

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-

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• •

•

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~ co

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6

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25 _

20

6

6

;- 15

4

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E

4

~ c

2

UJ

2

16

3-0-glucosylation were determined similarly. The relationship between reaction velocity and delphinidin concentration increased between 3 and 60 ,...M (Fig. 5). With UDP-glucose as a donor, the ap-

'2 14

J.s 10

i

12

8

18

E

~

12 16

Fig. 4. Effect of UDP-glucose concentration on glucosyltransferase activity. Assays were carried out as described in the standard conditions and UDP-glucose concentration was varied.

•

'2 14

-

0 4 8 1/[SJ (10'4)

UDp· Glucose concentration (10,3 M)

16

Q.

·4

20

16

C)

•

•

Ol

18

~

•

Q.

•

o

·0.8

10

20

30

40

.0.4 0 0 0.4 1/[SJ(1D')

50

08

60

12

70

Cyanidin concentration (10 -<;M)

Fig. 3. Effect of cyanidin concentration on glucosyltransferase activity. Assays were carried out as described in the standard conditions and cyanidin concentration was varied.

·04

0

o

10

20

30

40

50

0.0

04 0.8 1/[S](1D')

1.2

60

80

70

90

Delphinidin concentration ( 10-<;M)

Fig. 5. Effect of delphinidin concentration on glucosyltransferase activity. Assays were carried out as described in the standard conditions and delphinidin concentration was varied.

CB. Do et al. / Plant Science 112 (1995) 43-51 Table 3 Properties of the UDP.glucose:cyanidin 3·0·glucosyltrans. ferase in Vilis vini/era cell suspension cultures pH activity optimum a p/b Molecular size b Km cyanidin a Km delphinidina Km UDP.glucose a Stimulation by Ca 2+. Mg2+c

8.0 4.55 56 kDa 18 I'M 28 I'M 1.2 mM none

aDetermined with enzyme after chromatofocusing. bDetermined with enzyme after gel filtration. cDetermined with crude extract.

parent Km and Vmax for delphinidin were 28 I'M and 19 nkatlmg protein, respectively. Addition of divalent cations to the assay had no significant effect on enzyme activity (results not shown). Molecular weight and isoelectric point. Only one fraction collected from the Shodex Protein gel filtration column and corresponding to a molecular weight of about 53 kDa, contained glucosyltransferase activity. On SDS-PAGE, this fraction gave one major band corresponding to an apparent molecular weight of about 56 kDa and a minor band. This minor protein was also present in an adjacent fraction which did not possess glucosyltransferase activity. The minor protein was therefore deemed to be a contaminant. From a crude enzyme extract, a rough pI estimate of 4.5-4.7 was determined by measuring the glucosyltransferase activity in cut out portions of a I-mm thick isoelectric focusing gel. The gel filtration fraction tested with glucosyltransferase activity showed only one major silver nitrate stained protein band observed in the mini isolectric focusing gel at pH 4.55. Table 3 summarizes a number of properties of UDP-glucose:cyanidin 3-0glucosyltransferase from V. vinifera cell cultures determined with the partially purified enzyme after ion-exchange, chromatofocusing and gel filtration liquid chromatographies.

4. Discussion The paper describes an enzyme which catalyzes the glucosylation of the 3-hydroxyl group of an-

49

thocyanidin. The purification method used resulted in an enrichment of 75-fold. Many properties of the enzyme were similar to those reported for anthocyanidin 3-0-glucosyltransferase from other species. The end product of the enzymatic glucosylation of cyanidin by the enzyme extracted from cell culture of Vilis vinifera is cyanidin 3-0-monoglucoside. Other glucosylation products of cyanidin were not observed under these assay conditions. In other systems [8,12, 14,20,22] cyanidin was also only glucosylated to the monoglucoside. The pH optimum for cyanidin glucosyltransferase from Vilis vinifera cell cultures was 8.0. The pH optimum of other anthocyanin [8,12,14,22] and flavonoid [11,16] pathway glucosyltransferases are similar. The glucosyltransferase isolated from Vilis vinifera cells was highly specific to the position of the hydroxyl group to be glucosylated and the oxidation of the heterocyclic ring and was affected by substitution patterns in positions 3' and 5' in the B ring. The enzyme displayed highest activity with cyanidin (3' OH, 5' H) and delphinidin (3' OH, 5' OH) and then, in decreasing order, with pelargonidin (3' H, 5' H), peonidin (3' OCH3, 5' H) and malvidin (3' OCH3, 5' OCH3). The highest affinity towards cyanidin and delphinidin suggests that the enzyme may accept a hydrogen bond from the substrate's hydroxyl group in 3' . The slower activity observed with anthocyanidins methoxylated in 3' and 5' compared with the protonated substrate may be attributed to steric hindrance. An enzyme responsible for glucosylating cyanidin in red cabbage seedlings was reported and purified 50-fold [8]. This enzyme preparation was not specific for cyanidin but glucosylated a variety of flavonoid compounds containing 3-hydroxyl group. In cell cultures of Daucus carota, cyanidin 3glucosyltransferase was not specific with respect to the aglycone [14]. By contrast, the enzyme from V. vinifera cell cultures and the 90-fold purified cyanidin 3-0-glucosyltransferase from Silene dioica [20] did not catalyze the glucosylation of quercetin and kaemferol which differ from cyanidin and pelargonidin only in the level of oxidation of the heterocyclic ring. The differences in apparent substrate specificity among the

50

C.B. Do et al. / Plant Science 112 (/995) 43-51

glucosyltransferases may result from the combined activity of enzymes that are not easily isolated and purified. Flavonols, however, are not considered precursors in anthocyanin biosynthesis [4). Most properties of the glucosyltransferase from Vilis vinifera cell cultures are in accordance with those reported from the literature. The molecular size and isoelectric point are in the range of values reported for other plant glucosyltransferases [16,19). Although flavonoid O-glucosyltransferases have been shown to have two subunits of half the molecular size [11), results of SDS-PAGE suggest that the enzyme isolated from cell suspension of Vilis vinifera is a monomer. Highly-selected cell suspensions of Gamay Freaux grape contain, in decreasing order of importance, peonidin 3-p-coumaroylglucoside, malvidin 3-glucoside, peonidin 3-glucoside and cyanidin 3-glucoside [28). While peonidin-based anthocyanins are derived from cyanidin 3glucoside [4), malvidin-based anthocyanins arise from delphinidin 3-glucoside [29). Owing to its substrate specificity our glucosyltransferase would constitute a common starting point to both pathways. The lower affinity of the enzyme for the 3 I -O-methyl derivatives of cyanidin and delphinidin contributes to our understanding as to why glucosylation of cyanidin and delphinidin precedes their methylation.

Acknowledgements The helpful advice of Dr. Gulshan Arora on chromatofocusing chromatography is gratefully acknowledged. Work on the rough estimation of the isolectric point was skilfully carried out by Robert Raymond as part of his curriculum at the Biochemistry Department of the Universite du Quebec it Montreal.

References [I) S. Lofty. A. Fleuriet. T. Ramos and J.J. Macheix. Biosynthesis of phenolic compounds in Vitis vinifera cell suspension cultures: study on hydroxycinnamoyl CoA:ligase. Plant Cell Rep .• 8 (1989) 93-96. (2) C.B. Do and F. Cormier. Effect of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension. Plant Cell Rep .• 9 (1991) 500-504.

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