1146 N otes

Distinct Substrate Specificity of Dihydroflavonol Materials and Methods 4-Reductase from Flowers of Petunia hybrida Plant material G. Forkmann and B. Ruhnau The studies included lines, which lack 3'- Universität Tübingen, Institut für Biologie II, Lehrstuhl für and flavonoid 3',5'-hydroxylase (recessive htl and Genetik, Auf der Morgenstelle 28, D-7400 Tübingen, Bun­ desrepublik Deutschland hfl), and the commercial strains “Red Titan” and Z. Naturforsch. 42c, 1146-1148 (1987); “Blue Titan” (Benary, Hann. Münden, FRG) which received A ugust 19, 1987 possess 3'-hydroxylase activity (dominant Htl) and Anthocyanin Biosynthesis, Dihydroflavonol 4-Reductase, 3',5'-hydroxylase activity (dominant Hfl), respec­ -3,4-diols (leucoanthocyanindins), Substrate tively [5, 6], The lines were cultivated in a green­ Specificity, Petunia hybrida house. Dihydroflavonol 4-reductase from Petunia flowers cata­ lyzes the reduction of dihydroquercetin to leucocyanidin Chemicals and labelled substrates and, in particular, of dihydromyricetin to , whereas reduction of the simple dihydroflavonol dihydro- (-l-)-Dihydroflavonols and 4-coumaroyl-CoA were kaempferol to could not be observed. from our laboratory collection. Leucoanthocyanin­ This special substrate specificity of dihydroflavonol 4-re­ ductase is most probably the reason for the observations dins were kind gifts from W. Heller (Neuherberg, that delphinidin derivatives are the main end products of FRG) and L. Britsch (Freiburg, FRG). Labelled anthocyanin biosynthesis in Petunia flowers, whereas an- ( + )-dihydroflavonols (3.09 GBq/mmol) were thocyanins based on pelargonidin are rarely found and, if present, are only formed in very small amounts. prepared enzymatically from [14C]malonyl-CoA (Amersham-Buchler, Braunschweig, FRG) and 4- coumaroyl-CoA as described earlier [2]. Introduction Dihydroflavonol 4-reductase, which is involved in Enzyme preparation and enzyme assay anthocyanin biosynthesis, catalyzes the stereospecific The preparation of crude extracts from buds and conversion of ( + )-dihydroflavonols to flavan-3,4-cis- young flowers and the gel filtration of the extracts diols (leucoanthocyanindins). The latter compounds was performed as described [2] with the exception are the immediate precursors for the respective that glycerol was omitted. The standard enzyme as­ anthocyanidins (Fig. 1) [1, 2]. say contained in a total volume of 100 |xl: 0.03 nmol The enzymes from flowers of Matthiola [2], Calli- radioactive substrate (87 Bq), 500 nmol NADPH in stephus [3], Sinningia [4], Dianthus (Forkmann, un­ 20 [a1 water and 112 ^ g protein in 0.1 m Mcllvaine published) and Dahlia (Grisebach, personal com­ buffer, pH 6.8, with 2.8 mmol 2-mercaptoethanol. munication) were found to use dihydrokaempferol, Incubation was carried out for 30 min at 25 °C. The dihydroquercetin and dihydromyricetin as substrate reaction mixture was immediately extracted twice for the reduction reaction, to the respective leucoan- with ethyl acetate (80 |il, 80 [xl) and the extract thocyanidins. Dihydromyricetin is even reduced by chromatographed on cellulose plates (Schleicher & enzyme extracts from flowers which naturally lack Schüll, Dassel, FRG) with the solvent system delphinidin derivatives. The substrate specificity of chloroform/acetic acid/water (10:9:1). Because at dihydroflavonol 4-reductase seemed therefore to be least leucodelphinidin is poorly extracted from the not essentially influenced by the B-ring hydroxyla­ reaction mixture, 50 jil of the enzyme assay were also tion pattern of dihydroflavonols. Enzymatic studies directly spotted on a cellulose plate and separated as with Petunia flowers extracts revealed now, how­ described above. After chromatography the plates ever, that the dihydroflavonol 4-reductase of this were scanned for radioactivity. Radioactive zones plant exhibits a high substrate specificity with regard were stripped of [7] and counted in Aqualuma. to the B-ring substitution pattern. Analytical methods were performed as described earlier [2, 3].

Results and Discussion Reprint requests to Dr. G. Forkmann. Verlag der Zeitschrift für Naturforschung. D-7400 Tübingen Because the synthesis of labelled dihydroflavonols 0341 - 0382/87/0900 -1146 $01.30/0 with different B-ring hydroxylation pattern is expen- N otes 1147

Kaempferol ->■ Pelargonidin

Quercetin -► Cyanidin

OH OH

Myricetin -► Delphinidin

OH OH

Fig. 1. Enzymatic formation of and anthocyanidins, respectively, and of flavonols from dihydro- flavonols with different B-ring hydroxylation pattern in Petunia flowers. I = dihydrokaempferol; II = dihydroquercetin; III = dihydromyricetin; IV = leucopelargonidin; V = leucocyanidin; VI = leucodelphinidin. 1 = dihydroflavonol 4-reductase; 2 = flavonoid 3'-hydroxylase; 3 = flavonoid 3',5'-hydroxylase; 4 = flavonol synthase. ----- ► Main pathway; ...... ► m inor pathw ay. sive and time consuming, dihydrokaempferol as the tan”, which we normally use for the synthesis of simplest compound is generally used for the charac­ labelled . In fact, we could now demon­ terisation of the reductase reaction. This substrate is strate dihydroflavonol 4-reductase activity. With en­ also converted by flavonoid 3'- and flavonoid 3',5'- zyme preparations from flowers of “Red Titan” con­ hydroxylase with NADPH as co-factor (Fig. 1). In taining flavonoid 3'-hydroxylase activity, conversion Petunia, activity of these two hydroxylases can be of dihydrokaempferol to dihydroquercetin and to excluded by the use of a line with recessive alleles of some leucocyanidin was observed. And with flower the genes Htl and Hfl [5, 6], extracts from “Blue Titan”, which possess flavonoid When flower extracts of such a line were incubated 3',5'-hydroxylase activity, some dihydroquercetin, with [I4C]dihydrokaempferol in the presence of dihydromyricetin and, in particular, leucodelphini­ NADPH, no formation of the respective flavan-3,4- din were found to be formed from dihydrokaemp­ diol leucopelargonidin was observed. Dihydrofla­ ferol. But reduction of dihydrokaempferol to the re­ vonol 4-reductase activity could also not be demon­ spective flavan-3,4-diol leucopelargonidin was again strated by variation of the enzyme preparation and of not observed (Table I). Thus, dihydroquercetin and the assay conditions including different pH-values, dihydromyricetin but not dihydrokaempferol seems temperature and time of incubation and NADPH to be a suitable substrate for the reductase reaction. concentration. In order to confirm this assumption, we prepared Commercial strains often exhibit much higher ac­ labelled dihydroquercetin and dihydromyricetin and tivities for enzymes involved in flavonoid biosyn­ incubated each with NADPH and flower extract of thesis than genetically defined mutant lines. For “Red Titan”. As expected, dihydroquercetin was further studies we therefore prepared enzyme ex­ reduced to leucocyanidin and dihydromyricetin to tracts from the strains “Red Titan” and “Blue Ti­ leucodelphinidin. Again the reduction rate of di- 1148 Notes

Table I. Amount of products formed from dihydrofla- This special substrate specificity is in complete vonols by action of flavonoid 3'-hydroxylase or 3'.5'-hy- agreement with the observation that in the flowers of droxylase and dihydroflavonol 4-reductase. Petunia hybrida derivatives of kaempferol and Conversion rate [%] quercetin and, with regard to anthocyanins, delphini- Line Substrate of the substrate to din derivatives are the main endproducts of flavo­ DHQ DHM LPg LCy LDp noid biosynthesis [8, 9]. Higher amounts of cyanidin

“Red Titan” DHK 42.4 _ _ 5.8 _ derivatives were only formed in the absence of “Blue Titan” DHK 12.6 16.7 - - 41.5 flavonol synthase, which competes with dihydrofla­ “Red Titan” DHQ -- 13.5 - vonol 4-reductase for dihydroquercetin as common “Red Titan” DHM - - - 80.4 substrate (8). In the presence of both enzyme DHK = dihydrokaempferol; DHQ = dihydroquercetin; activities, dihydroquercetin is mainly converted to DHM = dihydromyricetin; LPg = leucopelargonidin; quercetin (Fig. 1). Such a competition does not oc­ LCy = leucocyanidin; LDp = leucodelphinidin. cur for dihydrokaempferol. In the presence of both flavonol synthase and dihydroflavonol 4-reductase activity, dihydrokaempferol is highly converted to hydromyricetin was considerably higher than that of kaempferol but not reduced to leucopelargonidin. In dihydroquercetin (Table I). Similar differences for flowers, which lack flavonol synthase or exhibit a the both substrates were found with enzyme extracts reduced activity, dihydrokaempferol is accumulated from flowers of “Blue Titan”. Moreover, with di­ (Fig. 1) [9], hydroquercetin and, in particular, dihydromyricetin From these results, it can be concluded, that the as substrate, dihydroflavonol 4-reductase activity substrate specificity of dihydroflavonol 4-reductase is could now also be demonstrated in flower extracts apparently the only reason for the rare occurrence from lines which lack flavonoid 3'- and flavonoid and, if present, for the small amounts of pelargonidin 3',5'-hydroxylase activity. derivatives in the flowers of this plant [10]. This con­ These results prove that, in contrast to the flower­ clusion is further confirmed by feeding experiments ing plants mentioned above, the dihydroflavonol 4- on flowers of Petunia, where leucopelargonidin was reductase from Petunia flowers exhibits a distinct found to be a suitable and strong precursor for the substrate specificity. Dihydromyricetin (3',4',5'- formation of pelargonidin glucosides (Forkmann, OH) is highly reduced to leucodelphinidin, whereas unpublished). dihydroquercetin (3',4'-OH) is only poorly con­ verted to leucocyanidin, and dihydrokaempferol (4'- Acknowledgements OH) is not used as substrate for the reduction reac­ These investigations were supported by grants tion (Fig. 1). from the Deutsche Forschungsgemeinschaft.

[1] W. Heller, L. Britsch, G. Forkmann, and H. [7] R. J. Redgwell, N. A. Turner, and R. L. Bieleski, J. Grisebach, Planta 163, 191 (1985). Chromatogr. 88 , 25 (1974). [2] W. Heller, G. Forkmann, L. Britsch, and H. [8] A. G. M. Geräts, P. de Vlaming, M. Doodeman, A. Grisebach, Planta 165, 284 (1985). Al, and A. W. Schram, Planta 155, 364 (1982). [3] B. R uhnau and G . Forkm ann, Phytochem istry, in [9] G. Forkmann, P. de Vlaming, R. Spribille, H. Wie- press (1988). ring, and A. W. Schram, Z. Naturforsch. 41c, 179 [4] K. Stich and G. Forkmann, Phytochemistry, in press (1986). (1988). [10] A. Cornu, M. Paynot, and H. Touvin. Phytochemistry [5] G. Stotz, Thesis, Tübingen 1983. 13, 2022 (1974). [6] G. Stotz, P. de Vlaming, H. Wiering, A. W. Schram, and G. Forkmann, Theor. Appl. Genet. 70, 300 (1985).