Characterization and activities of S-adenosyl-l -methionine:cyanidin 3-glucoside 3′-O-methyltransferase in relation to anthocyanin accumulation in Vitis vinifera cell suspension cultures

ELSEVIER

Plant

Science 122 (1997) 81-89

Characterization and activities of S-adenosyl-L-methionine:cyanidin 3-glucoside 3’-O-methyltransferase in relation to anthocyanin accumulation in Vitis vinzfera cell suspension cultures Christophe Food Rwarch

Bailly’, Franqois

Cormier*,

Chi Bao Do

and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Blvd West, St. Hyacinthe Qukbec J2S 8E3, Canada Received

17

July 1996; revised 25 October

1996; accepted

25 October

1996

Abstract

A cell suspension of Vitis vinifera cv Gamay Freaux was grown in a maintenance and an anthocyanin-promoting medium (APM). Time-course changes in anthocyanin accumulation and S-adenosyl-t-methionine:cyanidin 3-glucoside 3’-O-methyltransferase (CGMT) activity were examined throughout the growth cycle. An increase in anthocyanin accumulation mainly due to peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside i.e. 3’-methylated derivatives of cyanidin 3-glucoside (Cy3G), of cells grown in APM was observed. The higher activity of CGMT in cells grown in the APM might be responsible for the enhanced formation of 3’-methylated anthocyanins. CGMT isolated from cell suspension cultures has been purified approx. 35-fold with 3.4% enzyme activity recovery using anion exchange, chromatofocusing and gel filtration liquid chromatography. Mg 2+ ions strongly enhanced the reaction rate at a concentration of 5 mM. Optimal pH of CGMT ranged from 7.75 to 9.75. Michaelis-Menten kinetics were observed at pH 8.75 with respect to the substrates Cy3G and S-adenosyl-L-methionine with K,,, values of 199 and 18 PM respectively. S-adenosyl-L-homocysteine showed a non-competitive inhibition with an approx. K, value of 50 ,uM. Relative to Cy3G. the activity of CGMT was 13% with cyanidin. No activity was observed with delphinidin or cyanidin 3-p-coumaroylglucoside. Copyright Q 1997 Elsevier Science Ireland Ltd. Kqwords:

Anthocyanin biosynthesis; Methyltransferase: Purification

Vitis vinifera: Cell cultures: SAM:cyanidin

3-glucoside 3’-O-methyltransferase;

1. Introduction * Corresponding author. Fax: + 1 514 7738461; e-mail: [email protected] ’ Present adress: BIOCEM, Laboratoire de Technologie des Semences, Avenue de Bois I-Abbe, 49070 Beaucouze. France. 0167-9452/97/$17.00

Copyright

PI1 S0168-9452(96)04545-l

0 1997 Elsevier

Science

Ireland

In Vitis vinifera cell suspension, it was noted that induction of high anthocyanin production was obtained following an osmolality raise of the Ltd. All rights

reserved

x2

C. Badly et al.

: Plant Scicvwr 12-7 (1997) 81~-89

growth medium provoked by a high sugar concentration [1,2] and/or as a consequence of a low nitrate concentration of the culture medium [3,4]. Such anthocyanin-promoting conditions were shown to be detrimental to biomass proliferation and the main anthocyanin accumulated was peonidin 3-glucoside [3,5]. In cell lines selected over an extensive period, peonidin 3-glucoside accumulation was accompanied by that of peonidin 3-pcoumaroylglucoside [4]. These observations suggest that the increase in the formation of 3’-Omethylated anthocyanins in Vi/is vinzjku cell suspensions in the anthocyanin-promoting culture conditions might involve an anthocyanin methyltransferase as has been shown to occur in flowers of Petuniu hybridu [6]. However, cellular events involved in the intracellular anthocyanin accumulation still remain unclear and little is known about the enzymes responsible for the hydroxylation, glucosylation, methylation and acylation steps leading to the terminal anthocyanin pigment [7]. The first glucosylation step which occurs mostly in position 3, seems to play an important role in the stabilization of the anthocyanins [8]. In a previous study we have partially characterized a UDP-glucose:cyanidin 3-0-glucosyltransferase (CGT) from a cell suspension culture of Vitis vinij~ra cv Gamay Freaux [9]. Although CGT could glucosylate a variety of 3-hydroxy anthocyanidins, it favoured anthocyanidins that were hydroxylated in 3’ or in 3’ and 5’ positions. These results suggested that the glucosylation of anthocyanidins preceded their methylation. To confirm this hypothesis we have set out to characterize S-adenosyl-L-methionine:cyanidin 3-glucoside 3’-0-methyltransferase (CGMT) especially with regards to substrate specificity and to determine its role in the accumulation of 3’-O-methylated anthocyanins.

2. Material and methods 2.1. Crll cultwes Cell suspensions of grape (V. vinifera, var Gamay Frtaux, cell line # 15.1) were grown in a maintenance medium (MM) and routine transfers

were performed as described previously [l]. Experiments were performed by transferring 7day-old suspension cultures to an anthocyaninpromoting medium (APM). The APM had the same composition as the MM except for sucrose which was replaced by 280 mM glucose and nitrates which were reduced to one-quarter the normal amount, i.e. 6.25 mM [3]. 2.2. Anthoqunin Anthocyanin were performed 2.3. Protein

unulysis quantification as described

extruction

and HPLC analysis previously [l-5].

und enzyme pur$icution

23.1. Chemicals Cyanidin, delphinidin, cyanidin 3-glucoside and peonidin 3-glucoside were from Extrasynthese (France). Cyanidin 3-p -coumaroylglucoside was prepared from grape cell cultures by preparative HPLC as described elsewhere [lo]. The structure was confirmed by analytical HPLC according to , data reported by Lofty et al. [l l] and by matrix assisted laser desorption time of flight mass spectrometry (MALDITOF-MS) using a-cyano4hydroxy-cinnamic acid as the matrix. Sadenosyl-L-methionine and S-adenosyl-L-homocysteine were from Boehringer (Germany). All other chemicals were of analytical reagent grade. 23.2. Bujfers All buffers contained 10 mM polyethylene glyco1 3400, 20 mM Na-diethyldithiocarbamate, 2 mM dithiothreitol and 14 mM b-mercaptoethanol unless stated otherwise. 23.3. Methyltrunsj&use ussuy Different assays were carried out to determine the optimal conditions for enzyme activity. The standard reaction mixture contained, in a total volume of 200 pl, 50 mM Tris-HCl pH 8.75, 0.25 mM cyanidin 3-glucoside. 2.5 mM SAM, 5 mM MgClz and 60 p 1 enzyme extract ( 12 to 14 pg protein). The reaction was initiated by adding the enzyme. The mixtures were incubated for 10 and 20 min at 35°C. The reaction was terminated by adding 100 pl HCI (5%). The methylation product

C. BnillJ et ul.

Plmt

of cyanidin 3-glucoside was identified as peonidin ?-glucoside by co-chromatography with an authentic sample using a HPLC procedure described previously [5]. Time-course experiment was carried out in triplicate. Peaks were integrated for quantitative determination using peonidin 3-glucoside as calibration standard. Enzyme activity was defined as moles of peonidin 3-glucoside produced per second (katal) under the assay conditions.

2.3.4.1. Estraction. Protein extraction and partial purification using 30% ammonium sulphate were performed as described previously [9]. The protein solution was desalted on PD-10 column (Sephadex G-25, Pharmacia) equilibrated with 50 mM Na-phosphate pH 6.2. This constituted the desalted crude extract for enzyme purification.

_7..1.4.2.I. km -cJschmngr Clzroi71Lltogruph1.. A Pharmacia Biopilot column (Q Sepharose 35 1100) connected to a preparative HPLC (System Gold, Beckman. California. USA) was used for ion-exchange chromatography. The Biopilot column was equilibrated with 50 mM Na-phosphate pH 6.2. Portions of desalted crude extract containing 250-300 mg of protein were injected onto the column. The enzyme was eluted with a linear gradient of NaCl (0.771 M) in 50 mM Na-phosphatc (pH 6.2) at a flow rate of 3 mljmin. Collected fractions of eluent (3 ml) were immediately desalted using PD-10 column equilibrated with 25 mM BissTris-IHCl (pH 6.3) and methyltransfcrase activity was tested in each fraction. The desalted active fractions of several runs were pooled and concentrated about four-fold using Centriprep membrane (exclusion limit 30 kDa, Amicon Corp., Toronto, Canada) prior to chromatofocusing. .?..~.4.2.2. Cllronlut~?fbcztsing. Chromatofocusing using a Mono P (HR5/20) column (Pharmacia, Uppsala. Sweden) was performed as described previously [9]. Buffers used for column equilibration and elution contained only /I-mercaptoethanol as enzyme protector. The pH of the

Sc~iertcr I.?2 (1997) 81~ 89

83

eluting fractions (2 ml) was adjusted immediately to pH 7.5 by the addition of 0.5 ml 2 M Tris-HCl (pH 7.5) and Polybuffer was removed from fractions collected by passage through PD-10 column after equilibration with 100 mM Tris-HCl (pH 7.5). Fractions containing methyltransferase activity were stored at - 20°C in the presence of 10% glycerol. .?.3.4.-3.3. Grl j&Won. Gel filtration using a Bio-Sil TSK-250 column (Bio-Rad) was performed as described previously [9]. Elution buffer contained only I-mercaptoethanol as enzyme protector. Z.3.4.3. Protein assa~~.~. Protein content of the preparation was estimated using a protein assay kit (Bio-Rad, California, USA) with ;*-globulin as the calibration standard [ 121. -7.3.4.4. Properties of’ pur!‘firtl ~w~~wle. Partially purified enzyme extracts from chromatofocusing chromatography were used to determine the effects of pH, the enzyme kinetic and the substrate specificity. Assays were performed twice in duplicate. Enzyme purified by gel filtration was used to determine molecular weight. -7.3.4.4.1. l$ixt of’ pH. The effect of pH on enzyme activity was determined in 100 mM Kphosphate (pH 6.557.75) 100 mM TrissHCI (pH 8.0%8.75) and 100 mM glycine -NaOH (pH 9.0~10.0) buffers at 35°C. The appropriate buffer containing 10 mM polyethylene glycol 3400, 3 mM dithiothreitol. 20 mM Na-diethyldithiocarbamate and 14 mM P-mercaptoethanol was used and enzyme activity was assayed under standard conditions. 2.3.4.4.2 Substrute specljicitj,. Specificity of the methyltransferase was studied using various substrates i.e. cyanidin, delphinidin. cyanidin 3-glucoside and cyanidin 3-p-coumaroyl glucoside and enzyme activity was assayed under standard conditions. Enzyme activities were expressed as a percentage of the reaction for cyanidin 3-glucoside. -3.3.4.4.3. Ejjtcct of’ diudrnt iorzs. The effect of divalent ions on enzyme activity was estimated using different ions under chloride form (tinal concentration 1 or 5 mM) and enzyme activity

C. Bail!,) et al. 1 Plant Science

84

was assayed under standard conditions. Enzyme activities were expressed as a percentage of the reaction rate in the absence of divalent ions. 2.3.4.4.4. Enzyme kinetic. The enzyme solution was incubated with various concentrations of substrates yielding a range of final concentration from 0.01 to 5 mM for cyanidin 3-glucoside and 0.01 to 10 mM for SAM. For best precision in determining the K, value, only initial enzyme velocity values at substrate concentration ranging from 20 to 80% saturation were used. 2.3.4.4.5. Effect of S-adenosyl-L-homocysteine. The effect of S-adenosyl-L-homocysteine (SAH) on enzyme activity was estimated using various concentrations of SAH in a final concentration from 40 to 200 ,uM and enzyme activities were assayed under standard conditions. Enzyme activity was expressed as a percentage of the reaction in the absence of SAH. of molecular weight. 2.3.4.4.6. Estimation Molecular weight was determined by SDS-PAGE

122 (1997) 81-89

3.2. Enzyme

The purification yield was low because the enzyme is unstable. Indeed the methyltransferase lost up to 75% of its activity within 24 h at 4°C. It was therefore necessary to include several protectors in the buffers. When stored at - 20°C in the presence of 10% glycerol no loss in enzyme activity was observed. With regards to CGMT purification the initial ammonium sulfate cut allowed mainly the elimination of some proteins to which phenolic and anthocyanin compounds bind (Table 1). Residual anthocyanins were removed from the preparation by passage through PD 10 columns. The desalted protein extract was applied to an ion exchange Q Sepharose column and methyltransferase activity eluted at 0.8 M NaCl. The extract had to be

1600

[91. 2.4. Statistical

purljication

1600 -

A

1400 -

analysis

1200 -

Multiple comparisons between means were done with Duncan’s multiple range test using SAS software (SAS Institute, Cary, NC).

1000 ?CT 2

800 -

aa

600 -

3. Results 3.1. Anthocyanin

analvsis

and activity

of CGMT

E

1600

8 The cultivation of Vitis cells in the APM significantly (P < 0.05) enhanced the accumulation of total anthocyanins (Fig. 1). The overall anthocyanin increase (from the amount at inoculation) was more than three-fold. Increases in peonidin 3-glucoside and in peonidin 3-p-coumaroylglucoside were mainly responsible for the increase in total anthocyanins (Fig. 1B). CGMT activity of cells grown in APM was characterized by two maxima occurring at days 2 and 11 of culture (Fig. 2). The second maximum was almost three-times the maximum at day 9 in the MM. Except for days 3 and 4 of culture, CGMT activity was significantly (P < 0.05) higher in cells grown in the APM.

5

1200 1400

1

1000 600

c

600 400 200 0

I 0

2

4

Tim

6

6

10

12

I&!

Fig. 1. Anthocyanin composition of Vitis uinijtra cell suspensions during growth in a maintenance medium (A) and in an anthocyanin-promoting medium (B). Cyanidin 3-glucoside (0). malvidin monoglucoside ( A ), peonidin 3-p-coumaroylglucoside (A ), and peonidin 3-glucoside ( : 1).

C. Bail/y

et ul.

Plunt Shwc~t~ 12

(1997)

81 -XY

85

mM cyanidin 3-glucoside. The reaction rate was maximal above 0.75 mM SAM. The value of the K, for SAM was found to be 18 /l M.

The methyltransferase activity was reduced by the presence of SAH (Fig. 6). Maximal inhibition corresponding to a 75% loss of enzyme activity, was obtained with 120 /IM of SAH. Under the standard assay conditions a higher concentration of SAH did not further inhibit the enzyme activity and the apparent K, of the CGMT for SAH was about 50 ,LIM.

0

2

4

6

8

10

12

Time Cd) Fig. 2. Specific activity of S-adenosyl-L-methionine:cyanidin Sglucoside 3’-O-methyltransferase (CGMT) of Viti.s rv~iferu cell suspensions during the growth cycle in a maintenance medium (0) and in anthocyanin-promoting medium ((-: ).

immediately desalted to keep its activity. Further purification was obtained by chromatofocusing on Mono P column. Methyltransferase activity emerged as a single peak. At the chromatofocusing step, the enzyme peak was eluted at around pH 5.1. More than 96% of enzyme activity from crude extract was lost during purification. 3.3. Pmprrties

oj’ the CGMT

3.3.1. Ejjtct of’ pH Although the enzyme activity seemed to be maximal at pH 7.75 and 9.75, all activity mea surements between pH 7.25 and 9.75 were in the same Duncan grouping (Fig. 3). Therefore. the median point between 7.75 and 9.75 i.e. 8.75. was used for the enzyme assays. 3.3.2. Enqwe kirzetics The maximum reaction rate was obtained with 0.25 mM cyanidin 3-glucoside, in the presence of 5 mM SAM (Fig. 4). The apparent K, value of the CGMT as determined for cyanidin 3-glucoside at pH 8.75 and 35°C was 199 yM. Fig. 5 shows the effect of different SAM concentrations on the methyltransferase activity in the presence of 0.25

The influence of different divalent ions on the enzyme activity is shown in Table 2. Mg’ + ions strongly enhanced the reaction rate particularly at a concentration of 5mM which stimulated the CGMT activity by more than two and a half-fold. With the exception of Fe” ions, which had almost no effect on the enzyme activity, other ions tested were potent inhibitors of methyltransferase at the concentrations used. The addition of EDTA (1 mM) resulted in a complete loss of enzyme activity.

Various anthocyanins and aglycones were employed to evaluate the substrate specificity of the CGMT (Fig. 7). Cyanidin 3-glucoside. the most abundant substrate present in grape cells, was the most efficient substrate in cjrr~) for the purified enzyme (Table 3). Cyanidin 3-P-coumaroylglucoside which was reported to occur in the l4ti.s cell culture [ 111, was isolated and the identity was confirmed by mass spectrometry. The molecular ion at m/z 595 and the loss of 308 mass units corresponding to the loss of [I-coumaroylglucose were observed. No activity was detected with delphinidin and cyanidin 3-/I-coumaroylglucoside. The enzyme could methylate cyanidin at a relatively low rate. 3.3.6. Molrculur wright Only one fraction collected from the Bio-Sil gel filtration column and corresponding to a molecular weight of about 80 kDa. contained methyl-

86

C. Buill>a et ul. / Plunt Scienc~e IX’ (1997) 81-89

Table I Purification

of SAM:cyanidin

Purification

steps

Crude extract (NH&SO,+PDlO Anion exchange Chromato-focusing

Total

3-glucoside protein

3’-@methyhransferase Specific activity

(mg)

810 292 2.59 0.77

(pkatal,‘mg

Purification

protein)

(-fold)

T j

cultures

Recovery

I

Previously, we have shown that high sugar and low nitrate concentrations promote anthocyanin accumulation with emphasis on the methylated derivatives of cyanidin 3-glucoside [3,5]. In Vitis riniferu cell suspensions, the major derivatives of cyanidin 3-glucoside are peonidin 3-glucoside and

450

li’ris ritl!frrcrcell suspension

from

(%)

100 86.1 6.3 3.4

2.4 19.8 35.6

4. Discussion

^

isolated

9.0 21.5 178.5 320.5

transferase activity. On SDS-PAGE, this fraction gave one major band corresponding to an apparent molecular weight of about 80 kDa and a minor band of approx. 35 kDa. Table 4 summarizes a number of properties of CGMT from L’. t++u cell cultures that were determined with the partially purified enzyme after ion-exchange and chromatofocusing chromatographies.

500

(CGMT)

peonidin 3-p-coumaroylglucoside. The conversion of cyanidin 3-glucoside into peonidin 3-glucoside is carried out by CGMT. In this enzymatic step, S-adenosyl-L-methionine (SAM) is the methyl donor as for other anthocyanidin methyltranferases [6,13,14]. Results show that the higher CGMT activity might be responsible for the enanhanced accumulation of 3’-methylated thocyanins in cells grown in the APM. We report here for the first time the partial purification and characterization of a methyltransferase catalyzing the methylation of cyanidin 3-glucoside. The purification procedure was strongly hindered by the high instability of the Nevertheless the enzyme was purified enzyme. 35-fold with a 3.4% yield, which is comparable to the results of purification procedures obtained

400

/=-----'

400 B p

350

1

300

G 3 250 13 200

1 1’”

150

100

i1 6

,~-~~~r~ -

0 7

8

9

10

200

Cyanidin

400

_~~~_ 600

‘10

’

~~r~~_ _800

3.glucoside concentration

1000

(PM)

PH Fig. 3. pH optimum of S-adenosyl-L-methionine:cyanidin glucoside 3’-O-methyltransferase (CGMT) isolated from tinifera cell cultures.

3Vitis

Fig. 4. Effect of cydnidin 3-glucoside concentration on methyltransferase activity. Assays were carried out as described in the standard conditions and cyanidin 3-glucoside concentration varied.

Table 1 Effect of divalent ions on the enzyme SAM:cyanidin 3-glucoside 3’-O-methyltransferase from I ‘irk ~Ynifrrn cell suspension cultures

400

350 -c .g

l

300

Divalent

2 CL Ex 250 7s 2

None Mg’+

(I mM) Mg” (5 mM) Fe2’ (I mM) CL?’ (I mM) 2n2’ (I mM) Ca“ (I mM) Hg” (I mMj EDTA (I mM)

200

f -$’

150

; m 2

100

z, 5

ions

50

Relative

activity

activity ol (CGMT)

(“G of control)

100 210 265 95 5.9 5.9 II.5 0. I 0

0

SAM Concentration (mM) Fig. 5. Effect of S-adenosyl-r-methionine (SAM) concentralion on methyltrdnsferase activity. Assays were carried out as described in the standard conditions and concentration of SAM varied.

with other methyltransferases [15,16]. Only two protein bands were revealed by SDS-PAGE after gel filtration suggesting that the CGMT was very close to homogeneity after this step. Nevertheless.

8oI

I

0

SAH Cancentration (PM) Fig. 6. Effect of S-adenosyl-L-homocysteine (SAH) concentration on methyltransferase activity. Assays were carried out as described in the standard conditions and concentration of SAH varied.

the dramatic loss of enzyme activity did not allow us to characterize CGMT activity with this preparation. CGMT seemed to exhibit two pH optimum ot 7.75 and 9.0. However. activities between these pHs were not significantly different. Methyltransferases involved in flavonoid biosynthesis generally have their pH-activity optimum around pH 8.0 [17F 191 or at a higher value [15,20]. Jonsson et al. [14] have reported that anthocyanin methyltransferases from Peturlicr h~hir/u displayed two pH optimum, close to our values (7.‘7 and 8.5. 9.0). Similarly to other flavonoid methyltransferases [ 14,151, CGMT required Mg’~+ ions fog full activity. Other ions tested were inefficient in enhancing the reaction rate. In the absence of 5 mM Mg’the methyltransferase activity was nevertheless detectable. The requirement of a metal ion was confirmed by the strong inhibitory effect of EDTA on enzyme activity. S-adenosyl-Lhomocysteine. which is known to be a strong inhibitor of methyltransferase [ 15.2 I], also inhibited the methylation of cyanidin 3-glucoside by CGMT in Vitis rinifkrn cell cultures. Although this inhibition was not fully characterized. the data obtained indicate that CGMT activity was strongly inhibited by low concentration of SAH. Since the K, of SAH and the K,,, of SAM are same order of magnitude the inhibition by SAH seems to be non-competitive [ 141 as has been observed with methyltransferase from cell suspension cultures of Ciwr trrirtimm [?O]. CGMT

C. Badly et al. 1 Pht

88

showed a higher affinity for SAM than for cyanidin 3-glucoside. The high affinity of other methyltransferases for SAM has been observed [14,22], as well as the marked differences between the K,, values for SAM and for the substrate [23,24]. In contrast with anthocyanin methyltransferases from Petunia hybrida which accept acylated anthocyanins as substrate [14], the presence of a non-acylated glucosyl group in position 3 of

12-1 (1997) 81-89

Table 3 Substrate specificity of SAM:cyanidin 3-glucoside 3‘-Omethyltransferase (CGMT) from Vitis uiniftra cell suspension cultures Substrates

Relative

Cyanidin 3-glucoside Cyanidin 3-p-coumaroylglucoside Cyanidin Delphinidin

100 0

OH

Cyanidin OH

OH

Delphinidin OH

3-glucoside

pH activity

Fig. 7. Chemical Table 3.

of CGMT

substrates

13 0

optimum”

7.75-9.75

Molecular weightb (kDa) K, cyanidin glucoside” (apparent) K, SAM” (apparent) (pm) K, SAH” (apparent) (pm) Stimulation by Mg’+ * (mM)

3-p-coumaroylglucoside

structures

(‘X of control)

Table 4 Properties of SAM:cyanidin 3-glucoside 3’-O-methyltransferase (CGMT) from Vitis tGzi@ra cell suspension cultures

OH

Cyanidin

activity

the anthocyanin C ring was a sine qua non requirement for CGMT activity from Vitis uinijh cell cultures. CGMT molecular weight was found to be approx. 80 kDa. Anthocyanin methyltransferase from Petunia hybrida has a molecular weight of 50 kDa [14] while flavonoid methyltransferases have a molecular weight ranging from 48 kDa [25] to 111 kDa [20]. The results presented demonstrate the existence of a highly specific methyltransferase in Vitis tliniferu cell suspension culture which catalyses the transfer of the methyl group of S-adenosyl-L-methionine to the 3’-hydroxyl group of cyanidin 3-glucoside. The fact that the enzyme has a very low affinity for cyanidin and methylates neither cyanidin 3-p-coumaroylglucoside nor deiphinidin, makes it very likely that peonidin-based anthocyanins are derived from cyanidin 3-glucoside and that the glucosylation of cyanidin precedes its methylation.

OH

Cyanidin

Scimw

listed

in

.’ Determined ’ Determined

with enzyme with enzyme

(pm)

80 199 18 50 5

after chromatofocusing after gel filtration.

Acknowledgements The authors are grateful to Eric Giteau for his technical support and to Lawrence Hogge from the Plant Biotechnology Institute of the National Research Council of Canada (Saskatoon, Canada) for mass spectrometry analysis. Christophe Bailly has received a post-doctoral fellowship from the Ministbe de la Recherche et de la Technologie (France).

LIZI M.M. Bradford, A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. B&hem.. 72 (1976) 148-254. M.E.G. Aarsman. A.W. Schram and [I31 L.M.V. Jonsson. G.J.H. Bennink. Methylation of anthocyanins by cell-free extracts of flower buds of Pctmitr h~~hrido. Phytochemistry. 21 (1982) 2457 2459. M.E.G. Aarsman. J.E. Poulton and (141 L.M.V. Jonsson. of foul A.W. Schrdm. Properties and genetic control ot allin methylation methyltransfcrascs invohcd

[I51

References [II F. Cormier,

H.A. Crevier and C.B. Do, Effects of sucrose concentration on the accumulation of anthocyanins in grape ( Vitis rin!fkz) cell suspension. Can. J. Bet.. 68 (1990) 1822~1825. I4 C. B. Do and F. Cormier, Accumulation of anthocyanins enhanced by a high osmotic potential in grape ( Vitis riGf&-a L.) cell suspensions. Plant Cell Report. 9 ( 1990) 143 146. of peonidin VI C. B. Do and F. Cormier, Accumulation 3-glucoside enhanced by osmotic stress in grape (Fitis cbtifka L.) cell suspension. Plant Cell Tissue Org. Cult.. 74 (1991) 49-54. proVI F. Cormier. C.B. Do and Y. Nicolas. Anthocyanin duction in selected cell lines of grape ( Vitis rini/>ra L.). In Vitro Cell. Dev. Biol., 30P (1994) 171-173. [51 C.B. Do and F. Cormier. Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape I Vi/is cin[fera L.) cell suspension. Plant Cell Report, 9 (1991) 500-504. [hl L.M.V. Jonsson. P. De Vlaming. H. Wiering, M .E.G. Aarsman and A.W. Schram. Genetic control 01‘ anthocyanin-O-methyltransferase activity in flowers of Pctuniu lt~hrida. Theor. Appl. Genet.. 66 (1983) 349 -355. [71 J.J. Macheix. A. Fleuriet and J. Billet. The main phenolies of fruit. in: Fruit Phenolic. CRC Press Inc., Boca Raton, Florida, 1990. pp. l--98. anthocyanins. CRC Crit. PI H.J. Francis, Food colorants: Rev. Food Sci. Nutr.. 28 (1989) 273-314. and Y. Nicolas. Isolation and 191 C.B. Do. F. Cormier characterization of a UDP-glucose:cyanidin 3-O-glucosyltransferase from grape cell suspension cultures ( l’itk vinifkra L.). Plant Sci., I I2 (1995) 43-51. F. Cormier, C.B. Do and R.R. [ 1(‘I M.R. Van Calsteren. Laing. ‘H and “C NMR assignements of the major anthocyanins from l’itis ainifhra cell suspension culture. Spectroscopy. 9 ( 199 I ) I 15. [I II S. Lofty, A. Fleuriet. T. Ramos and J.J. Macheix. Blosynthesis of phenolic compounds in Vitis cinijtra cell suspension culture: study on hydroxycinnamoyl CoA:ligase. Plant Cell Rep., 8 (1989) 93-96.

[I61

[I71

[I81

[I91

PO1

PII

[22]

[?3]

P41

[15]

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