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Identification, properties and genetic control of hydroxycinnamoyl-coenzyme A: Anthocyanidin. 3-rhamnosyl (1 → 6) glucoside, 4‴-hydroxycinnamoyl transfersae isolated from petals of Silene dioica
Biochem. Physiol. Pflanzen 175, 403-411 (1980)
Identification, Properties and Genetic Control of Hydroxycinnamoyl-coenzyme A: Anthocyanidin 3-rhamnosyl (1 ---76) glucoside, 4'/1 -hydroxycinnamoyl Transfersae Isolated from Pet als of Silene dioica1 ) JOHN KAl\ISTEEG, JAN VAN BREDERODE, CEES H. HOMMELS and GERRlT VAN NlGTEVECHT Department of Population and Evolutionary Biology, University of Utreeht, The Netherlands K ey Term In d ex: eyanidin- und pelargonidin-glyeosides, acyl-anthocyanins, acyltransferase, anthotyanin biosynthesis, genetic control; SilcI/I] diolea.
Summary An enzyme eat1tlyzing the transfer of the p-coumaroyl or caffeoyl moiety of respeetively p-coumaroyl-CoA and ('affeoyl-CoA to the 4-hydroxyl grollp of the rhamnosyl moiety of anthocyanidin ß-rhamnosyl(l -~ G)glu('osides and ß-rhamosyl(l -->- G)glncoside-5-glucosides has been demonstrated in petal extraets of Silelle diolen plants. This aeyltransferase aetivity is governed by gene Ac j in petals of ac/ac plants this a(·tivity is 15-times lower than in petals of Ac! Ac plants. The enzyme purified fiftyfold by Dowex 1 x 2 anr! Sephadex G-150 ehromatography, exhibits a pH optimum of 7.G -- 7.8, has a Illolecular wei ght of 5(;,000 daltons, is not sti Illillated by divalent metal ions, and has a "true Km" value of 3.5/Ür for caffcoyl-CoA, and of 0.29 mM for cyanidin ß-rhamnosyl(l -->- 6)glucosirle. p-Counmroyl-CoA ('an ,t1su serve as substrate uOllur ("true Km" 15,u:VI). Fur the acceptor pelargonidin ß-rhamllosyl(l -+ G)glncoside a "trne Km" of O.Oß m:\1 has been obtained.
Introduction
In reddish-purple flowers of European populations of Silene dioica cyanidin 3rhamnosyl (1 _ 6) glucoside-5-glucoside and its acylated derivative cyanidin 3-(4-caffeoylrhamnosyl (1 _ 6)glucoside )-5-g1ucoside in a 1: 9 ratio are always found (KAMSTEEG ct al. 1976, 1978a). Crosses with a S. dioica mutant with no acyl-anthocyanins in the petals (K.UISTEEG et al. 1976) showed that Olle single dominant gene Ac controls acylation. In pink flowcrs of a mutant of S. dioica, originallyfound in Limburg, The Netherlands, pelargonidin 3-rlMmnosyl (1 _ 6)glucoside-5-g1ucoside and its acylated derivative pelargonidin ß-(4-p-coumaroylrhaJUllosyl(1_ 6)glucoside)-5-g1ucoside are present in a 1: 9 ratio. Crosses with 8. dioica clemonstrated that gene P controls not only the hydro1) This illvestigatioll w,tS snpported by a grant from the research poul of the University of Utreeht.
404
J. KAMSTEEG et al.
xylation of pelargonidin to cyanidin (and therefore flower colour), but also that of the acyl moiety p-coumaric acid to caffeic acid. Biochemical studies (KAMSTEEG et al. 1980a) revealed that gene P actually governs the formation of the enzyme p-coumaroylCoA, 3-hydroxylase, which catalyzes the conversion of p-coumaroyl-CoA to caffeoylCoA. In p/p plants which cannot convert p-coumaroyl-CoA, the condensation of p-coumaroyl-CoA with three acetate units of malonyl-CoA finally leads to the formation of pelargonidin. In these plants p-coumaroyl-CoA is also used as substrate in the acylation step. This explains why in p/p plants pelargonidin-glycosides are acylated with p-coumaric acid. The enzyme which catctlyzes the condensation of the hydroxycinnamoyl-CoA ester with malonyl-CoA (chalcone-flavanone sYllthetase) has, however, a much higher affinityand V max for caffeoyl-CoA than for p-coumaroyl-CoA. Therefore only cyanidin-glycosides are present in petals of P/P plants. But the question remains why in these plants in which both p-coumaroyl-CoA and cctffeoyl-CoA should be present, only caffeoyl-CoA is used for the acylation step, as up to now no cyanidin-glycosides acylated with p-coumaric acid have been detected. To elucidate this problem, the properties and genetic control of the enzyme which catalyzes the acylation step have been studied. Material and Methods Plant Material The growing eonditions and harvesting of plant material have been described before (VAN NIGTEVECIIT 1966). Thc genotype mim. NI N lacks anthocyanidin 3-rhamnosyl(1 -+ 6)glllcoside, 5-0·glucosyltransferase aetivity (KAMSTEEG et a1. 1976; 1978; 1979b).
Chenricals UDP-D·(U-14C)glucose (300 Ci/Mol) was supplied by the Radiochemieal Centre Amersham, England; (ß_14C) malonic acid (46 Ci/Mol) by NEC'f Corp., Boston, Massaehusetts. (ß_14C) p-Coumaric acid and (ß_14C) caffeic acid were synthesized from (ß_14C) malonie aeid and p-hydroxybenzaldehy:le or 3,4-dihydroxybenzaldehyde, respeetively, according to the Knoevenagel condensation lIsing pyridine and a traee of piperidine for eahtlysis. p-Collmaroyl-CoA was a gift of Dr. R. SÜTFELD, Prof. M. R. ZENK amI Dr. B. ULllJ1[('If; caffeoyl-CoA was a gift of Prof. Dr. 1\1. H. ZEi'\K and Dr. B. ULBIUCIl. Cyanidin-glyeosides were isolated from petals of appropriate genotypes of 8. diaica as described befoTe (KAMSTEEG et a1. 1978 cl. Pelargonidin 3-0-glucoside was isolated from strawberries lIecording to the method of WnOLSTAD ct 111. (lmO).
EnzYlIw Prcparatir)// All operlltions were ("lIrried out IIt 0-4 °C. Five grams of petals were homogenllted in IIn a11 glass Potter Elvehjem homogenizer in 10 m!, 20 mM ß-mcrcllptoethanol, 5% soluble polyvinylpyrrolidone, 1°!c) Triton X-100, 50 mM potllssium-sodillm phosphate buffer (pR 7.6), lind centrifllged for 15 min at 38,000 . g. Thc protein was purified from phenolics and other low moleeuillf weight substllnees by filtration over a Dowex 1 x 2 (0.8 by 10 em) lind a Sephadex G-50 (2.5 by 30 ('111) eolumn, subsequently. The eO!llmns had been cqllilibrated befofe llse with a 4 mM ß-mercllptoethanol, 10 mM potassillm-sodium phosphate buffer (pH 7.G). This fmction was routineollsly llsed in the investigations of the enzyme propcrties.
Hydroxycinnamoyl-Coenzyme A
405
Assays of enzyme Activily Assa/! lII ethod 1.: syuthesis of ((Ilthocyallidiu -3( U_H C)rhamnosy 1)-(1 -+ 6)glucoside by pre-incubatioll. The pre-in('lIbation reaetion mixtllre contained in a total vohun e of 60 !ll: 48 nmoles UDP-L[U-14C]rhamnose (;) Ci/:\!ol) (or 0.:14 nmol es LTDP- D-[lptC]gItH'ose (300 Ci/moles) and I!unol NADPH), 0.5 flllloles potassillm-sodillm phospluLte buffer (plI 7.6), 0.:2 !lInolcs ß-Illereaptoethanol and 50 rtl enzymc of a gCllotypc la('kin g ii-O-g-Ill('osyltrallsferase artivity (m/m N/N Ac/"1c). The reaction mixture was imllbated for 40 min at ,lO "C amI stoppe
Results
Conditions for enzyme actiüity and enzyme properties Incubations of UDP- D-gl ucose, labelIed uuifo1'mly in the glucose moiety, with cyanidin 3-rhamuosyJ(1 -+ 6)glu cosie!e and the SUpel'llatant of a cmde homogenate of petals of Silene dioica, 1'es ulted not onl)' in ineo1'poration of 1'adioaetivity in cyanidin 3-rhamuosyl(1 -+ ß)glucoside-5-glucoside, but also in cyallidin 3-( 4-caffeoyl1'hamllosyl(1 _ 6)glucoside-5-g1ueosidc (KA:\ISTEEG et al. 1978 b). \Vhen t hc enzyme was omittcd from thr re action lllixture, 01' trichlo1'oacetic acid was added befo1'c incllbation, no inco1'poration of radioaetivity was observed. It has been dcscribce! bcfol'r (KAMSTEEG ct a1. 1978 b) that thn cllzYIllr UDP-D-glucose: anthoeyanidill 3-rhamnosyl(1 -+ 6)gllleoside, 5-0-g1ueosyltransfnasl' catalyzes thn trallBfer of the glucosyl moiety from UDP-D-glucose to the 5-hyd1'oxyl g]'oup of a,nthocyanins. Becau se the 5-0-glueosyltransfc1'ase cannot g lucos:datp ('ltdogl'oOUS eyanidin 3-( 4-eaffeoylrhalllnosyl(1 -;>- ü)gluco side) (KAJlISTEEG, unpublishrd resu lts), after 5-0-glucosylation, anothcl' enzyme has to cataIyzc the attachmellt of tl1<' l'lldOgl'lleOUS caffeic acid to t he 4-hydroxyl group of the rhanlllosylllloiety of eya 11 idin 3-1'hamnosyl(1 --'7 6)glucosidc-5-(U-14C)glueosie!e. When the sUjlel'llatallt of the emde homogenate was passee! through a Polyclar AT (PVP) alld d Sf'phadex G-50 columll, subsequelltly it has lost it8 aeylating activity almost completf'ly.Howevcr, \Vhell the supcrnatallt was passee! through a Dowex 1 x 2and a Sephadex G-öO eolulIlll it was still able to synthes ize aeyl-anthocyanins, 28 Biochcm. Physiol. Pflanzen, Ud.liü
J.
406
KAlIISTEEG
et al.
although at a rcduced rate. Even after Sephadex G-150 chromatography, some of the protein fractions still formed cyanidin 3-( 4-caffeoylrhamnosyl(1 -.)0 6)glucoside)-5-(U-14C)glucoside from cyanidin 3-rhamnosyl(1 -.)0 6)glucoside and UDP-D-(U_14 C)glucose, indicating that the acylating enzyme probably elutes together with its substrate (Table 1). Table 1. Purification of H!fdroxyci/1/lI1l1loyl-Coellz!fme A: anthocyanidin 3-rhamnosyl(1 4'" -hydroxycinnanwyltralls{erasc isolated {rom pe/als of 8. dioicll
-+
6 )glucoside,
Protein fraetion
Vohllne (mI)
Protein (mg/mI)
Total activity (unitsl/min)
Homogenized erude extraet Snpernatant crnde extmet Dowex 1 x 2 eluate Sephadex G-50 eluate Sephadex Cl-150 eluate
15.2
6.;)
19.76
0.20
1
L3.2
4.G
17.01
0.28
1.4
11.G
1.07
97.14
7.79
38.95
18.7
0.59
92.35
8.37
41.85
1;).;)
0.ß9
G2.89
10.54
52.7
Speeific aetivity (unitsjminjmg prot.)
Enzyme purity
one nnit is the amollnt of enzyme which catalyzes the formation of 1 nm()l acylanthoeyanin per minute at 30 oe in the assay method 1.
1
Addition of caffeic acid to the above mentioned assay hardly led to any stimulation in acyl-anthocyanin formation. A fourfold stimulation, however, was found when the substrates, necessary for the formation of caffeoyl-CoA in S. dioica (KAMSTEEG, unpublishedresults): caffeic acid, ATP. CoASH, and M'gCI 2 , M'nC1 2 or CaClz, were addcd to the assay. This suggests that caffeoyl-CoA is thc hydroxycinnamoyl donor. To denlOl1strate the involvcment of caffeoyl-CoA, [ß_14C] labelIed caffeic acid M'gCI 2, CoASH, ATP and unlabelled cyanidin 3-rhaumosyl(1 -.)0 6) glucosidc were incubated with the supernatant of a crude petal homogenate. Thc formed 14C-Iabclled anthocyanin, cyan in 3-( 4-caffeoylrhamnosyl(1 ~ 6)glucoside was purified and subjected to mild alkaline hydrolysis as described before (KAJ\iISTEEG et a1. 1978a). The acyl compound was extracted with diethyl ether alld after concentration applied, together with various references on silica gel GF 2;4 thin layer plates aud developed in thc solvent systems benzene-p-dioxane-aceticacid(90: 25: 4, v/v/v) (PASTUSKA 1969). The water phase was applied on Whatman 111 anel developeel in ßAW anel 15 (10 acetic aeiel respectivcly. The radioactivity cochromatographed only with citffeic acid; no radioactivity was found on the deacylated anthocyanin spot. Also after incubation of [ß_14C] labelIed caffeoyl-CoA and cyanidin 3-rhamnosyl(1 -.)0 6)glucoside with a protein preparation of petals of Ac/ Ac plants, 14C-Iabelled cyanidin 3-(4-caffeoylrhamnosyl(1 -.)0 6)glucosiele) was formed. Because of the difficulties encountered with the synthesis of [ß_14C] caffcoyl-CoA with beef liver butyryl CoA-ligase (HOJlDiELS and KAJlfSTEEG, unpublished results),
Hydroxyeinnamoyl-Coenzyme A
407
only part of the experiments were performed this way. Labelling the anthocyanin acepctor is another possibility. Here difficulties are met during purifieation and concentration. Anthocyanins degradate easily and are only stable in acid solutions. This problem could be circumvented by synthesizing 14C-labelled anthocyanin in pre-incubation experiments. Incubation mixtures prepared in parallel wem used to determine the amount of 14C-Iabelled anthocyanin formed during pre-incubation.
Protein and Time Linearity The amount of acyl-allthocyanin formed from cyanidin 3-rhamnosyl(1 -+ 6)glucoside-5-g1ucoside was found to be proportional to added enzyme and to time for periode up to 5-8 min.
pH Optimum The acyltrallsferase exhibited a pR optimum of 7.G-7.8. The same pR optimum
(± 0.2 unit) was found with the different anthocyanin substrates.
lnfluence of divalent metallons and other Agents Although EDTA (pH 7.5) inhibited the reaction rate, none of the divalent metal
ions tested (Ca 2+, C0 2+, Mg2+, Mn 2+ and Zn 2+) stimulated the re action (Table II). The enzyme was irreversibl.r inhibited by HgCI 2 , strongly inhibited by N-ethylmaleimide, but only slightly by p-chloromereuribenzoatc (Table 2). Both cysteine and ß-mercaptoethan)l could counteract the inhibitiol'i in the presence of N-ethylmaleimide and p-chloromercuribenzoate. Table 2. R{ted ot rli 1'11 11'/1 t 11/11111 ;01/8 U/ld sulfltydryl 8jJecific reagents Oll hydroxycillllllJiloyl-Coenzyme A: anthocyau/dill 3-rlt'UllI/o8IJI( 1 --+ 6 )glur:oside, 4'" -hydroJ:ycin/lIl!lloyltra}/sfernse. The readioJ\ Illixtllre was lIssa,yed lIS desnibe,J in ~Iaterial an,[ Methods (assay IJ)cth:l
COlllpound
Final ('onl'entration (111:\1)
Pcn:ent maximal aetivity
1
GI 41 GO 37
ETWA CaCl 2 CoCl z
40
1
:\IgCI 2
:\InCI 2
1:3
ZnCl 2
III'CI,
1
p-rhloro l11ercuri bCllzoate N-ethylmaleimide
1.~5*
97
1.25*
1:2
.)
}VIolecular wwight Determination The molecular weight of the acyltransferase was detcrmined according to the method of ANDREWS (19G5) using a Sephadex G-150 column. The column was calibrated with 28*
408
J.
KA MS TEEG
et
011.
cytochrome-c, chymotrypsinogen A, egg albumin, aldol ase, and catalase as standards and as eluting buffer a 10 mM potassium-sodium phosphate solution containing 4 mM ß-mercaptoethanol (pR 7.5). On t his Sephadex G-150 column the acylating activity eluted together with the 5-0-g1ucosyltransferase activity in a fraction corresponding to a molecular wcight of approxim at ely 56,000 daltons. That the 5-0-g1ucosyltransferase is not involved in the acylation of anthocyanins can be concluded from the finding that protc ill preparations of petals of mim N/N Ac/Ac S. dioica plants, lacking 5-0-glucosyltransferase (KAM:STEEG et al. 1978 b), also possess maximal acylating activity in that fractioll. Evell without thc addition of hydroxycinnamoyl-CoA ester, this fraction was still able to acylate anthocyal1ins, although at a strongly reduced rate. This suggests t hat the hydroxy-cilluamoyl moiety is also present in this fractiol1.
Substrate Specificity The acyltransferase exhibits a rather broad substrate specificity both in "vitro and in vivo. In petals of 8. dioica with the genotype M/ M n/n Ac/ Ac, which contain a mixture of cyal1idin 3-glucoside, 3,5-diglucoside al1d 3-(6-caffcoylglucoside )-5-glucoside, the acyl moiety is bound to the ti-hydroxyl group of the 3-0-boul1d glucose. I n vitro, protein extracts of petals of M / M n/n Ac/ Ac plants were also able to acylate the 4-hydroxyl group of the rhamnose moiety of anthocyanidil1 3-rhamnosyl(1 -+ 6)glucoside when this anthocyanil1 was offered as su bstrate. In addition, a protein preparation of petals of S. dioica plants, which contain cyanidin 3-(4-caffcoylrhamnosyl(1 - ;. 6)glucoside)-5-glucoside (M/M N/N Ac/Ac), was also abl e to acylate the 6-hydroxyl group of the 3-0-bound glucose of cyanidin 3,5-diglucoside. Also \vith regard to the acyl moiety, the substrate specificity is rather broad, both p-coumaroyl-CoA and caffeoyl-CoA can be used as acyl donor (Table 3). Table 3. Tme Jfic/uwlis J.~lc llt en Const!/.l1ts nnd F max o( hydroxycillnamoyl-Coenzymc Li: nnthocya.nidin 3-rlt amnosyl(1 --+ 6 )glucosidc, 4'" -lIcyltmnsfemse for various substrates Sub stmte cyanillin 3-rhamllosylglllcoside caffeoyl-Co A cyanillin il-rhamllosylglllcoside p -conmaroyl-CoA pelargonid in 3-rhalllllosylglllcosidc p -ro lllllaroy l- CoA
True i\li chaelis co nstant (lll :\t ) 0.29 0.0035
O.;}O 0.013
o.m
O.OLl
r mf x (nlllol flllinfmg protein)
35
24
45
Kinetics For the determination of tho various killctic parameters, the assay method 1 was used. Different concelltrations of pelargonidin- or cyanidin 3-([U-14 C]rhamnosyl)-( 1 -+ G)glucoside were otained by varying either the incubatiol1 time, 01' the concelltratiOll of the anthocyanin acceptor in thc pre-incubation. The final concentration of the allthocyanidin 3-([lP4C]rhamno syl)( -(1 -+ 6)glucosides was determined by preparillg
l-Iydroxycinnamoyl-Coenzyme A
409
incubation mixtures in parallel. Tbc pre-incubation was stopped with 1 mM p-chloromercuribenzoate, which inhibited the rhamnosylation reaction for 97 % (KAMSTEEG et al. 1980a). p-Chloromercuribenzoate was alm ost without cffect on the acylating reaction (3 % inhibition only). Lineweaver-Burk plots (LINEWEAVER and BURK 1934) of the reciprocal of the initial velocity of the acylation reaction versus the reciprocal of the hydroxycinnamoyl-CoA ester concentration at different fixed cyanidin 3-rhamnosyl(1 -+ 6)-glucoside concentrat ions showed a linear relationship between I /V and l /hydroxycinnamoyl-CoA concentration, thc apparent Km being dependcnt upon the second substrate (anthocyanin) concentration. When the reciprocal of the initial velocity is plotted against the l'eciprocal of thc anthocyanin concentration at several different fixed concentrations of hydroxycinnamoyl-CoA esters, the same linear relationship was found. Secondary plots, according to FLORINI and VESTLING (1957), of the intcrcepts on the I/V axis versus the l/concentration of the fixed substrate. These kinetic parameters are given in Table 3. From this table it can be concluded that thn enzyme has a higher affinity for caffeoyl-CoA than for p-coumaroyl-CoA, but also has a higher affinityfor pelargonidin-glycosides than for cyanidin-glycosides.
Genetic Contl'ol Data in Table 4 show that the presence of the dominant alleles of gene Ac is necessary for optimal production of the enzyme that catalyzes the transfer of the caffeoyl moiety of caffeoyl-CoA to the 4-hydroxyl group of rhamnose of the 3-0- bound rutinose of anthoTable 4. Activity or h!Jdrox!Jcl:nnamoyl-Coe1tz.I/llw A: flnthoc!Janidin 3-rltamnosyl(1 -)- 6 )glucoside, 4'" -ltlfdroxycillltnllloyltrrntsfcl'IIse in jletnls or vlIrions genot!Jpus (lf 8. dioi ca. Abbreviations user!: ey, eyanidin; l'g, pelargonidin; R, rhamnose ; G, glucose; ac, acylated. 'fhe 4'" -h ydroxyeinnamoyltransfcrase aetivity was ass;tyed as desl'fibed in Materials amI :vr ethods cpm Co- chroma,tographetl with carrier cYltnidin ß-(4-caffcoylrhamnosyl(1 -+ G)glucoside) Genotype
Anthoeyanin ]lfCsent
4'" -hydroxycinnamoyl-transferase aetivity (epm)*
P-mm N- A.cp- mm N- acac pp mm N- AcP- M- im Ae-
ey BRG-
ac
GOO
Pg :1RG- ac
547
ey
41
3RG
ey :mG/3G 5G/
3G-ae5G
5:38.
cyanidin 3-rhamnosyl(1 -+ 6)glucosides. Hardly any acyltransfersae activity was found in petals of ac/ac plants. From this it may be concludcd that the hydroxycinnamoylCoA: anthocyanidin 3-rhamnosyl(1 -+ 6)glucoside 4" '-hydroxycinnamoyltransferase exhibits the properties expected of an acyltransferase which is controlled by gene Ac. The gene was also present in petals of p/p M/M N/N Ac/Ac plants, which contain pelal'gonidin 3-(4-p-coumaroylrhamnosyl(1 _ 6)glucoside)-5-g1ucoside and pelargonidin 3-rhamnosyl(1 -+ 6)glucoside-5-glucoside. The acyltransferase activity, determined in the presence of cyanidin 3-rhamnosyl(1 -+ 6)glucoside and caffeoyl-CoA as substrates,
410
J. KA MSTEEG ct al.
was in petals of pjp plants as high as in petals of Pj P plants. This demonstrates that the action of gene Ac is independent of that of gene P, which indirectly controls the hydroxylatiOll pattern of the acyl moiety transferred.
Discussion
In this article we describe an enzyme which catalyzes the acylation of the 4-hydroxyl group of rhamllose of anthocyanidin 3-rhamnosyl(1 -+ 6)glucosides. This acyltransferase has the following properties : (i) only 3-0-bound glycosides can be acylated. (ii) In vivo the acylation in petals of S. dioica is only complete (> 90%) with anthocyanidin 3-rhamnosyl(1 -+ 6)glucosides or 3-rhamnosyl(1 -+ 6)glucoside-5-glucosides. In this case the acyl group is bound to the 4-hydroxyl group of the rhamnose moiety(KAMsTEEG et al. 1978a). When the acyl group is bound at the 6-hydroxyl group of the 3-0-bound glucose, e.g. with anthocyanidin 3-glucosides or 3,5-diglucosides, in vivo never more than 20% of total anthocyanin is acylated. (iii) The acyltransferse can use both p-coumaroyl-CoA and caffeoyl-CoA as p-coumaroyl, respectively caffeoyl donor. It has, however, a higher affinity for caffeoyl- CoA than for p-coumaroyl-CoA, but has also a higher affinity for pelargonidin than for cyanidin-glycosides. For the various substrates the Vmax did not differ very much. From the finding that anthocyanidin 3-( 4-hydroxycinnamoylrhamnosyl(1 -+ 6)glucoside are no appropriate substrates for the 5-0-g1ucosyltransferase it may be concluded that acylation is a final step in anthocyanin biosynthesis. Until now wo failed to liberate detectable amounts of p-coumaric acid after alkaline hydrolysis of acylated canidin 3-rhamnosyl(1 -+ 6)glucoside-5-g1ucoside from petals of P/P plallts. The question why cyanidin-glycosides are solely acylated with caffeic acid cannot be completely solvcd. It is difficult to accept that t he difference in affinity for the hydroxycinnamoyl-CoA esters alol1c cal1 explain this co-occurrel1ce. Abettel' explanation is that the conversion of p-coumaro yl-CoA to caffeoyl-CoA, controlled by gene P is complete, so in petals of PjP plants only caffeoyl-CoA is available for the acylation step. Acknowledgements The authors are much indeb ted to Prof. Dr. M. H. ZE N K amI Dr. B. ULBl\ICH, Lehrstuhl für Pflanzenphysiologie, Ruhr- Universität, Boehllm, FRG, and Dr. R. SÜTFELD, Botanis ches Institut der Westfälischen Wilhelms-Univcrsitiit, Mü nster, FRG, for kindl y snpplying sa mpi es of p -coum aroy l- CoA and caffeoyl-CoA.
References ANDREWS, P.: Thc gel-filtration behaviour of pro teins related to their molecuhu wcights over a wide ra nge. Biochem. J. 96, 595 - 606 (1965) . FLORINl, J . R., and VESTLlN G, C. S.: Graphieal determination of the dissoeiation constants for two substrate enzyme systems. Biochim. Biophys. Acta 25, 575 -578 (1957).
Hydroxycinnamoyl-Coenzyme A
411
I\.<\.MS'l'BBG, J., BHEDBROD E, J. V,I N, and :'-IlGTEVIlCHT, G. VAN: Pleiotropic effett oI a pelargonidinh ydroxy l;Ltion gene in Si/elle dinicI!? Phytodlemistry 15, 1917 -1918 (197G). - Ki;PPlms, F. J. E. l\f. , a1HI NIGTBI' ECIlT, G. VI:>: Anthocyanins isolated from petals of various genotypes of the Red Campioll (Sil ene dioicu (L.) Cillirv.). Z. Natllrforsl'h. 33C, 475-483 (1978a). - and NIGTEVE('HT, G. VAN: Jdentifi eatioll, properties and genet il' control of CDP-gillcose: cyanidiu-3-rhanmosyl-(1 -~ G)glll('oside-5-0-glucosyltransferase isolated from petals of the Red Campion (8ill'll(, dioiC/l). Hioehem. Genet . 16, 1059-1071 (1978b). - - Identifil'ation aud properties of UD P-gllleose: cyanidin-3-0-glllcosyltmnsferase isolated frorn petals of thc He
Receired Deeember 27, 1979. Authors' address: .lOH N KA~lSTJ; EG, Department of Pupulat ion and Evolutionary ßiulogy, University of Utrecht, Padn alaa n 8, Utree ht, 'rhe Netherlands.
Identification, Properties and Genetic Control of Hydroxycinnamoyl-coenzyme A: Anthocyanidin 3-rhamnosyl (1 ---76) glucoside, 4'/1 -hydroxycinnamoyl Transfersae Isolated from Pet als of Silene dioica1 ) JOHN KAl\ISTEEG, JAN VAN BREDERODE, CEES H. HOMMELS and GERRlT VAN NlGTEVECHT Department of Population and Evolutionary Biology, University of Utreeht, The Netherlands K ey Term In d ex: eyanidin- und pelargonidin-glyeosides, acyl-anthocyanins, acyltransferase, anthotyanin biosynthesis, genetic control; SilcI/I] diolea.
Summary An enzyme eat1tlyzing the transfer of the p-coumaroyl or caffeoyl moiety of respeetively p-coumaroyl-CoA and ('affeoyl-CoA to the 4-hydroxyl grollp of the rhamnosyl moiety of anthocyanidin ß-rhamnosyl(l -~ G)glu('osides and ß-rhamosyl(l -->- G)glncoside-5-glucosides has been demonstrated in petal extraets of Silelle diolen plants. This aeyltransferase aetivity is governed by gene Ac j in petals of ac/ac plants this a(·tivity is 15-times lower than in petals of Ac! Ac plants. The enzyme purified fiftyfold by Dowex 1 x 2 anr! Sephadex G-150 ehromatography, exhibits a pH optimum of 7.G -- 7.8, has a Illolecular wei ght of 5(;,000 daltons, is not sti Illillated by divalent metal ions, and has a "true Km" value of 3.5/Ür for caffcoyl-CoA, and of 0.29 mM for cyanidin ß-rhamnosyl(l -->- 6)glucosirle. p-Counmroyl-CoA ('an ,t1su serve as substrate uOllur ("true Km" 15,u:VI). Fur the acceptor pelargonidin ß-rhamllosyl(l -+ G)glncoside a "trne Km" of O.Oß m:\1 has been obtained.
Introduction
In reddish-purple flowers of European populations of Silene dioica cyanidin 3rhamnosyl (1 _ 6) glucoside-5-glucoside and its acylated derivative cyanidin 3-(4-caffeoylrhamnosyl (1 _ 6)glucoside )-5-g1ucoside in a 1: 9 ratio are always found (KAMSTEEG ct al. 1976, 1978a). Crosses with a S. dioica mutant with no acyl-anthocyanins in the petals (K.UISTEEG et al. 1976) showed that Olle single dominant gene Ac controls acylation. In pink flowcrs of a mutant of S. dioica, originallyfound in Limburg, The Netherlands, pelargonidin 3-rlMmnosyl (1 _ 6)glucoside-5-g1ucoside and its acylated derivative pelargonidin ß-(4-p-coumaroylrhaJUllosyl(1_ 6)glucoside)-5-g1ucoside are present in a 1: 9 ratio. Crosses with 8. dioica clemonstrated that gene P controls not only the hydro1) This illvestigatioll w,tS snpported by a grant from the research poul of the University of Utreeht.
404
J. KAMSTEEG et al.
xylation of pelargonidin to cyanidin (and therefore flower colour), but also that of the acyl moiety p-coumaric acid to caffeic acid. Biochemical studies (KAMSTEEG et al. 1980a) revealed that gene P actually governs the formation of the enzyme p-coumaroylCoA, 3-hydroxylase, which catalyzes the conversion of p-coumaroyl-CoA to caffeoylCoA. In p/p plants which cannot convert p-coumaroyl-CoA, the condensation of p-coumaroyl-CoA with three acetate units of malonyl-CoA finally leads to the formation of pelargonidin. In these plants p-coumaroyl-CoA is also used as substrate in the acylation step. This explains why in p/p plants pelargonidin-glycosides are acylated with p-coumaric acid. The enzyme which catctlyzes the condensation of the hydroxycinnamoyl-CoA ester with malonyl-CoA (chalcone-flavanone sYllthetase) has, however, a much higher affinityand V max for caffeoyl-CoA than for p-coumaroyl-CoA. Therefore only cyanidin-glycosides are present in petals of P/P plants. But the question remains why in these plants in which both p-coumaroyl-CoA and cctffeoyl-CoA should be present, only caffeoyl-CoA is used for the acylation step, as up to now no cyanidin-glycosides acylated with p-coumaric acid have been detected. To elucidate this problem, the properties and genetic control of the enzyme which catalyzes the acylation step have been studied. Material and Methods Plant Material The growing eonditions and harvesting of plant material have been described before (VAN NIGTEVECIIT 1966). Thc genotype mim. NI N lacks anthocyanidin 3-rhamnosyl(1 -+ 6)glllcoside, 5-0·glucosyltransferase aetivity (KAMSTEEG et a1. 1976; 1978; 1979b).
Chenricals UDP-D·(U-14C)glucose (300 Ci/Mol) was supplied by the Radiochemieal Centre Amersham, England; (ß_14C) malonic acid (46 Ci/Mol) by NEC'f Corp., Boston, Massaehusetts. (ß_14C) p-Coumaric acid and (ß_14C) caffeic acid were synthesized from (ß_14C) malonie aeid and p-hydroxybenzaldehy:le or 3,4-dihydroxybenzaldehyde, respeetively, according to the Knoevenagel condensation lIsing pyridine and a traee of piperidine for eahtlysis. p-Collmaroyl-CoA was a gift of Dr. R. SÜTFELD, Prof. M. R. ZENK amI Dr. B. ULllJ1[('If; caffeoyl-CoA was a gift of Prof. Dr. 1\1. H. ZEi'\K and Dr. B. ULBIUCIl. Cyanidin-glyeosides were isolated from petals of appropriate genotypes of 8. diaica as described befoTe (KAMSTEEG et a1. 1978 cl. Pelargonidin 3-0-glucoside was isolated from strawberries lIecording to the method of WnOLSTAD ct 111. (lmO).
EnzYlIw Prcparatir)// All operlltions were ("lIrried out IIt 0-4 °C. Five grams of petals were homogenllted in IIn a11 glass Potter Elvehjem homogenizer in 10 m!, 20 mM ß-mcrcllptoethanol, 5% soluble polyvinylpyrrolidone, 1°!c) Triton X-100, 50 mM potllssium-sodillm phosphate buffer (pR 7.6), lind centrifllged for 15 min at 38,000 . g. Thc protein was purified from phenolics and other low moleeuillf weight substllnees by filtration over a Dowex 1 x 2 (0.8 by 10 em) lind a Sephadex G-50 (2.5 by 30 ('111) eolumn, subsequently. The eO!llmns had been cqllilibrated befofe llse with a 4 mM ß-mercllptoethanol, 10 mM potassillm-sodium phosphate buffer (pH 7.G). This fmction was routineollsly llsed in the investigations of the enzyme propcrties.
Hydroxycinnamoyl-Coenzyme A
405
Assays of enzyme Activily Assa/! lII ethod 1.: syuthesis of ((Ilthocyallidiu -3( U_H C)rhamnosy 1)-(1 -+ 6)glucoside by pre-incubatioll. The pre-in('lIbation reaetion mixtllre contained in a total vohun e of 60 !ll: 48 nmoles UDP-L[U-14C]rhamnose (;) Ci/:\!ol) (or 0.:14 nmol es LTDP- D-[lptC]gItH'ose (300 Ci/moles) and I!unol NADPH), 0.5 flllloles potassillm-sodillm phospluLte buffer (plI 7.6), 0.:2 !lInolcs ß-Illereaptoethanol and 50 rtl enzymc of a gCllotypc la('kin g ii-O-g-Ill('osyltrallsferase artivity (m/m N/N Ac/"1c). The reaction mixture was imllbated for 40 min at ,lO "C amI stoppe
Results
Conditions for enzyme actiüity and enzyme properties Incubations of UDP- D-gl ucose, labelIed uuifo1'mly in the glucose moiety, with cyanidin 3-rhamuosyJ(1 -+ 6)glu cosie!e and the SUpel'llatant of a cmde homogenate of petals of Silene dioica, 1'es ulted not onl)' in ineo1'poration of 1'adioaetivity in cyanidin 3-rhamuosyl(1 -+ ß)glucoside-5-glucoside, but also in cyallidin 3-( 4-caffeoyl1'hamllosyl(1 _ 6)glucoside-5-g1ueosidc (KA:\ISTEEG et al. 1978 b). \Vhen t hc enzyme was omittcd from thr re action lllixture, 01' trichlo1'oacetic acid was added befo1'c incllbation, no inco1'poration of radioaetivity was observed. It has been dcscribce! bcfol'r (KAMSTEEG ct a1. 1978 b) that thn cllzYIllr UDP-D-glucose: anthoeyanidill 3-rhamnosyl(1 -+ 6)gllleoside, 5-0-g1ueosyltransfnasl' catalyzes thn trallBfer of the glucosyl moiety from UDP-D-glucose to the 5-hyd1'oxyl g]'oup of a,nthocyanins. Becau se the 5-0-glueosyltransfc1'ase cannot g lucos:datp ('ltdogl'oOUS eyanidin 3-( 4-eaffeoylrhalllnosyl(1 -;>- ü)gluco side) (KAJlISTEEG, unpublishrd resu lts), after 5-0-glucosylation, anothcl' enzyme has to cataIyzc the attachmellt of tl1<' l'lldOgl'lleOUS caffeic acid to t he 4-hydroxyl group of the rhanlllosylllloiety of eya 11 idin 3-1'hamnosyl(1 --'7 6)glucosidc-5-(U-14C)glueosie!e. When the sUjlel'llatallt of the emde homogenate was passee! through a Polyclar AT (PVP) alld d Sf'phadex G-50 columll, subsequelltly it has lost it8 aeylating activity almost completf'ly.Howevcr, \Vhell the supcrnatallt was passee! through a Dowex 1 x 2and a Sephadex G-öO eolulIlll it was still able to synthes ize aeyl-anthocyanins, 28 Biochcm. Physiol. Pflanzen, Ud.liü
J.
406
KAlIISTEEG
et al.
although at a rcduced rate. Even after Sephadex G-150 chromatography, some of the protein fractions still formed cyanidin 3-( 4-caffeoylrhamnosyl(1 -.)0 6)glucoside)-5-(U-14C)glucoside from cyanidin 3-rhamnosyl(1 -.)0 6)glucoside and UDP-D-(U_14 C)glucose, indicating that the acylating enzyme probably elutes together with its substrate (Table 1). Table 1. Purification of H!fdroxyci/1/lI1l1loyl-Coellz!fme A: anthocyanidin 3-rhamnosyl(1 4'" -hydroxycinnanwyltralls{erasc isolated {rom pe/als of 8. dioicll
-+
6 )glucoside,
Protein fraetion
Vohllne (mI)
Protein (mg/mI)
Total activity (unitsl/min)
Homogenized erude extraet Snpernatant crnde extmet Dowex 1 x 2 eluate Sephadex G-50 eluate Sephadex Cl-150 eluate
15.2
6.;)
19.76
0.20
1
L3.2
4.G
17.01
0.28
1.4
11.G
1.07
97.14
7.79
38.95
18.7
0.59
92.35
8.37
41.85
1;).;)
0.ß9
G2.89
10.54
52.7
Speeific aetivity (unitsjminjmg prot.)
Enzyme purity
one nnit is the amollnt of enzyme which catalyzes the formation of 1 nm()l acylanthoeyanin per minute at 30 oe in the assay method 1.
1
Addition of caffeic acid to the above mentioned assay hardly led to any stimulation in acyl-anthocyanin formation. A fourfold stimulation, however, was found when the substrates, necessary for the formation of caffeoyl-CoA in S. dioica (KAMSTEEG, unpublishedresults): caffeic acid, ATP. CoASH, and M'gCI 2 , M'nC1 2 or CaClz, were addcd to the assay. This suggests that caffeoyl-CoA is thc hydroxycinnamoyl donor. To denlOl1strate the involvcment of caffeoyl-CoA, [ß_14C] labelIed caffeic acid M'gCI 2, CoASH, ATP and unlabelled cyanidin 3-rhaumosyl(1 -.)0 6) glucosidc were incubated with the supernatant of a crude petal homogenate. Thc formed 14C-Iabclled anthocyanin, cyan in 3-( 4-caffeoylrhamnosyl(1 ~ 6)glucoside was purified and subjected to mild alkaline hydrolysis as described before (KAJ\iISTEEG et a1. 1978a). The acyl compound was extracted with diethyl ether alld after concentration applied, together with various references on silica gel GF 2;4 thin layer plates aud developed in thc solvent systems benzene-p-dioxane-aceticacid(90: 25: 4, v/v/v) (PASTUSKA 1969). The water phase was applied on Whatman 111 anel developeel in ßAW anel 15 (10 acetic aeiel respectivcly. The radioactivity cochromatographed only with citffeic acid; no radioactivity was found on the deacylated anthocyanin spot. Also after incubation of [ß_14C] labelIed caffeoyl-CoA and cyanidin 3-rhamnosyl(1 -.)0 6)glucoside with a protein preparation of petals of Ac/ Ac plants, 14C-Iabelled cyanidin 3-(4-caffeoylrhamnosyl(1 -.)0 6)glucosiele) was formed. Because of the difficulties encountered with the synthesis of [ß_14C] caffcoyl-CoA with beef liver butyryl CoA-ligase (HOJlDiELS and KAJlfSTEEG, unpublished results),
Hydroxyeinnamoyl-Coenzyme A
407
only part of the experiments were performed this way. Labelling the anthocyanin acepctor is another possibility. Here difficulties are met during purifieation and concentration. Anthocyanins degradate easily and are only stable in acid solutions. This problem could be circumvented by synthesizing 14C-labelled anthocyanin in pre-incubation experiments. Incubation mixtures prepared in parallel wem used to determine the amount of 14C-Iabelled anthocyanin formed during pre-incubation.
Protein and Time Linearity The amount of acyl-allthocyanin formed from cyanidin 3-rhamnosyl(1 -+ 6)glucoside-5-g1ucoside was found to be proportional to added enzyme and to time for periode up to 5-8 min.
pH Optimum The acyltrallsferase exhibited a pR optimum of 7.G-7.8. The same pR optimum
(± 0.2 unit) was found with the different anthocyanin substrates.
lnfluence of divalent metallons and other Agents Although EDTA (pH 7.5) inhibited the reaction rate, none of the divalent metal
ions tested (Ca 2+, C0 2+, Mg2+, Mn 2+ and Zn 2+) stimulated the re action (Table II). The enzyme was irreversibl.r inhibited by HgCI 2 , strongly inhibited by N-ethylmaleimide, but only slightly by p-chloromereuribenzoatc (Table 2). Both cysteine and ß-mercaptoethan)l could counteract the inhibitiol'i in the presence of N-ethylmaleimide and p-chloromercuribenzoate. Table 2. R{ted ot rli 1'11 11'/1 t 11/11111 ;01/8 U/ld sulfltydryl 8jJecific reagents Oll hydroxycillllllJiloyl-Coenzyme A: anthocyau/dill 3-rlt'UllI/o8IJI( 1 --+ 6 )glur:oside, 4'" -hydroJ:ycin/lIl!lloyltra}/sfernse. The readioJ\ Illixtllre was lIssa,yed lIS desnibe,J in ~Iaterial an,[ Methods (assay IJ)cth:l
COlllpound
Final ('onl'entration (111:\1)
Pcn:ent maximal aetivity
1
GI 41 GO 37
ETWA CaCl 2 CoCl z
40
1
:\IgCI 2
:\InCI 2
1:3
ZnCl 2
III'CI,
1
p-rhloro l11ercuri bCllzoate N-ethylmaleimide
1.~5*
97
1.25*
1:2
.)
}VIolecular wwight Determination The molecular weight of the acyltransferase was detcrmined according to the method of ANDREWS (19G5) using a Sephadex G-150 column. The column was calibrated with 28*
408
J.
KA MS TEEG
et
011.
cytochrome-c, chymotrypsinogen A, egg albumin, aldol ase, and catalase as standards and as eluting buffer a 10 mM potassium-sodium phosphate solution containing 4 mM ß-mercaptoethanol (pR 7.5). On t his Sephadex G-150 column the acylating activity eluted together with the 5-0-g1ucosyltransferase activity in a fraction corresponding to a molecular wcight of approxim at ely 56,000 daltons. That the 5-0-g1ucosyltransferase is not involved in the acylation of anthocyanins can be concluded from the finding that protc ill preparations of petals of mim N/N Ac/Ac S. dioica plants, lacking 5-0-glucosyltransferase (KAM:STEEG et al. 1978 b), also possess maximal acylating activity in that fractioll. Evell without thc addition of hydroxycinnamoyl-CoA ester, this fraction was still able to acylate anthocyal1ins, although at a strongly reduced rate. This suggests t hat the hydroxy-cilluamoyl moiety is also present in this fractiol1.
Substrate Specificity The acyltransferase exhibits a rather broad substrate specificity both in "vitro and in vivo. In petals of 8. dioica with the genotype M/ M n/n Ac/ Ac, which contain a mixture of cyal1idin 3-glucoside, 3,5-diglucoside al1d 3-(6-caffcoylglucoside )-5-glucoside, the acyl moiety is bound to the ti-hydroxyl group of the 3-0-boul1d glucose. I n vitro, protein extracts of petals of M / M n/n Ac/ Ac plants were also able to acylate the 4-hydroxyl group of the rhamnose moiety of anthocyanidil1 3-rhamnosyl(1 -+ 6)glucoside when this anthocyanil1 was offered as su bstrate. In addition, a protein preparation of petals of S. dioica plants, which contain cyanidin 3-(4-caffcoylrhamnosyl(1 - ;. 6)glucoside)-5-glucoside (M/M N/N Ac/Ac), was also abl e to acylate the 6-hydroxyl group of the 3-0-bound glucose of cyanidin 3,5-diglucoside. Also \vith regard to the acyl moiety, the substrate specificity is rather broad, both p-coumaroyl-CoA and caffeoyl-CoA can be used as acyl donor (Table 3). Table 3. Tme Jfic/uwlis J.~lc llt en Const!/.l1ts nnd F max o( hydroxycillnamoyl-Coenzymc Li: nnthocya.nidin 3-rlt amnosyl(1 --+ 6 )glucosidc, 4'" -lIcyltmnsfemse for various substrates Sub stmte cyanillin 3-rhamllosylglllcoside caffeoyl-Co A cyanillin il-rhamllosylglllcoside p -conmaroyl-CoA pelargonid in 3-rhalllllosylglllcosidc p -ro lllllaroy l- CoA
True i\li chaelis co nstant (lll :\t ) 0.29 0.0035
O.;}O 0.013
o.m
O.OLl
r mf x (nlllol flllinfmg protein)
35
24
45
Kinetics For the determination of tho various killctic parameters, the assay method 1 was used. Different concelltrations of pelargonidin- or cyanidin 3-([U-14 C]rhamnosyl)-( 1 -+ G)glucoside were otained by varying either the incubatiol1 time, 01' the concelltratiOll of the anthocyanin acceptor in thc pre-incubation. The final concentration of the allthocyanidin 3-([lP4C]rhamno syl)( -(1 -+ 6)glucosides was determined by preparillg
l-Iydroxycinnamoyl-Coenzyme A
409
incubation mixtures in parallel. Tbc pre-incubation was stopped with 1 mM p-chloromercuribenzoate, which inhibited the rhamnosylation reaction for 97 % (KAMSTEEG et al. 1980a). p-Chloromercuribenzoate was alm ost without cffect on the acylating reaction (3 % inhibition only). Lineweaver-Burk plots (LINEWEAVER and BURK 1934) of the reciprocal of the initial velocity of the acylation reaction versus the reciprocal of the hydroxycinnamoyl-CoA ester concentration at different fixed cyanidin 3-rhamnosyl(1 -+ 6)-glucoside concentrat ions showed a linear relationship between I /V and l /hydroxycinnamoyl-CoA concentration, thc apparent Km being dependcnt upon the second substrate (anthocyanin) concentration. When the reciprocal of the initial velocity is plotted against the l'eciprocal of thc anthocyanin concentration at several different fixed concentrations of hydroxycinnamoyl-CoA esters, the same linear relationship was found. Secondary plots, according to FLORINI and VESTLING (1957), of the intcrcepts on the I/V axis versus the l/concentration of the fixed substrate. These kinetic parameters are given in Table 3. From this table it can be concluded that thn enzyme has a higher affinity for caffeoyl-CoA than for p-coumaroyl-CoA, but also has a higher affinityfor pelargonidin-glycosides than for cyanidin-glycosides.
Genetic Contl'ol Data in Table 4 show that the presence of the dominant alleles of gene Ac is necessary for optimal production of the enzyme that catalyzes the transfer of the caffeoyl moiety of caffeoyl-CoA to the 4-hydroxyl group of rhamnose of the 3-0- bound rutinose of anthoTable 4. Activity or h!Jdrox!Jcl:nnamoyl-Coe1tz.I/llw A: flnthoc!Janidin 3-rltamnosyl(1 -)- 6 )glucoside, 4'" -ltlfdroxycillltnllloyltrrntsfcl'IIse in jletnls or vlIrions genot!Jpus (lf 8. dioi ca. Abbreviations user!: ey, eyanidin; l'g, pelargonidin; R, rhamnose ; G, glucose; ac, acylated. 'fhe 4'" -h ydroxyeinnamoyltransfcrase aetivity was ass;tyed as desl'fibed in Materials amI :vr ethods cpm Co- chroma,tographetl with carrier cYltnidin ß-(4-caffcoylrhamnosyl(1 -+ G)glucoside) Genotype
Anthoeyanin ]lfCsent
4'" -hydroxycinnamoyl-transferase aetivity (epm)*
P-mm N- A.cp- mm N- acac pp mm N- AcP- M- im Ae-
ey BRG-
ac
GOO
Pg :1RG- ac
547
ey
41
3RG
ey :mG/3G 5G/
3G-ae5G
5:38.
cyanidin 3-rhamnosyl(1 -+ 6)glucosides. Hardly any acyltransfersae activity was found in petals of ac/ac plants. From this it may be concludcd that the hydroxycinnamoylCoA: anthocyanidin 3-rhamnosyl(1 -+ 6)glucoside 4" '-hydroxycinnamoyltransferase exhibits the properties expected of an acyltransferase which is controlled by gene Ac. The gene was also present in petals of p/p M/M N/N Ac/Ac plants, which contain pelal'gonidin 3-(4-p-coumaroylrhamnosyl(1 _ 6)glucoside)-5-g1ucoside and pelargonidin 3-rhamnosyl(1 -+ 6)glucoside-5-glucoside. The acyltransferase activity, determined in the presence of cyanidin 3-rhamnosyl(1 -+ 6)glucoside and caffeoyl-CoA as substrates,
410
J. KA MSTEEG ct al.
was in petals of pjp plants as high as in petals of Pj P plants. This demonstrates that the action of gene Ac is independent of that of gene P, which indirectly controls the hydroxylatiOll pattern of the acyl moiety transferred.
Discussion
In this article we describe an enzyme which catalyzes the acylation of the 4-hydroxyl group of rhamllose of anthocyanidin 3-rhamnosyl(1 -+ 6)glucosides. This acyltransferase has the following properties : (i) only 3-0-bound glycosides can be acylated. (ii) In vivo the acylation in petals of S. dioica is only complete (> 90%) with anthocyanidin 3-rhamnosyl(1 -+ 6)glucosides or 3-rhamnosyl(1 -+ 6)glucoside-5-glucosides. In this case the acyl group is bound to the 4-hydroxyl group of the rhamnose moiety(KAMsTEEG et al. 1978a). When the acyl group is bound at the 6-hydroxyl group of the 3-0-bound glucose, e.g. with anthocyanidin 3-glucosides or 3,5-diglucosides, in vivo never more than 20% of total anthocyanin is acylated. (iii) The acyltransferse can use both p-coumaroyl-CoA and caffeoyl-CoA as p-coumaroyl, respectively caffeoyl donor. It has, however, a higher affinity for caffeoyl- CoA than for p-coumaroyl-CoA, but has also a higher affinity for pelargonidin than for cyanidin-glycosides. For the various substrates the Vmax did not differ very much. From the finding that anthocyanidin 3-( 4-hydroxycinnamoylrhamnosyl(1 -+ 6)glucoside are no appropriate substrates for the 5-0-g1ucosyltransferase it may be concluded that acylation is a final step in anthocyanin biosynthesis. Until now wo failed to liberate detectable amounts of p-coumaric acid after alkaline hydrolysis of acylated canidin 3-rhamnosyl(1 -+ 6)glucoside-5-g1ucoside from petals of P/P plallts. The question why cyanidin-glycosides are solely acylated with caffeic acid cannot be completely solvcd. It is difficult to accept that t he difference in affinity for the hydroxycinnamoyl-CoA esters alol1c cal1 explain this co-occurrel1ce. Abettel' explanation is that the conversion of p-coumaro yl-CoA to caffeoyl-CoA, controlled by gene P is complete, so in petals of PjP plants only caffeoyl-CoA is available for the acylation step. Acknowledgements The authors are much indeb ted to Prof. Dr. M. H. ZE N K amI Dr. B. ULBl\ICH, Lehrstuhl für Pflanzenphysiologie, Ruhr- Universität, Boehllm, FRG, and Dr. R. SÜTFELD, Botanis ches Institut der Westfälischen Wilhelms-Univcrsitiit, Mü nster, FRG, for kindl y snpplying sa mpi es of p -coum aroy l- CoA and caffeoyl-CoA.
References ANDREWS, P.: Thc gel-filtration behaviour of pro teins related to their molecuhu wcights over a wide ra nge. Biochem. J. 96, 595 - 606 (1965) . FLORINl, J . R., and VESTLlN G, C. S.: Graphieal determination of the dissoeiation constants for two substrate enzyme systems. Biochim. Biophys. Acta 25, 575 -578 (1957).
Hydroxycinnamoyl-Coenzyme A
411
I\.<\.MS'l'BBG, J., BHEDBROD E, J. V,I N, and :'-IlGTEVIlCHT, G. VAN: Pleiotropic effett oI a pelargonidinh ydroxy l;Ltion gene in Si/elle dinicI!? Phytodlemistry 15, 1917 -1918 (197G). - Ki;PPlms, F. J. E. l\f. , a1HI NIGTBI' ECIlT, G. VI:>: Anthocyanins isolated from petals of various genotypes of the Red Campioll (Sil ene dioicu (L.) Cillirv.). Z. Natllrforsl'h. 33C, 475-483 (1978a). - and NIGTEVE('HT, G. VAN: Jdentifi eatioll, properties and genet il' control of CDP-gillcose: cyanidiu-3-rhanmosyl-(1 -~ G)glll('oside-5-0-glucosyltransferase isolated from petals of the Red Campion (8ill'll(, dioiC/l). Hioehem. Genet . 16, 1059-1071 (1978b). - - Identifil'ation aud properties of UD P-gllleose: cyanidin-3-0-glllcosyltmnsferase isolated frorn petals of thc He
Receired Deeember 27, 1979. Authors' address: .lOH N KA~lSTJ; EG, Department of Pupulat ion and Evolutionary ßiulogy, University of Utrecht, Padn alaa n 8, Utree ht, 'rhe Netherlands.