Biochemical and Ontogenetic Evidence that the Ferulic Acid and Isoscoparin Formation in Silene are Catalyzed by Different Enzymes

Abstract

Summary In this paper the ontogeny, biosynthesis and the regulation by light of the 6-C-glucosyl-flavones, isoorientin and isoscoparin is described. The total amount present of these compounds is strongly influenced by the light level at which these plants are raised. Only at high light levels and in the later periods of development, the contribution of these substances to the total flavone content becomes of importance. In the early stages and in the petals they are absent. Enzymatic studies on the ontogeny of the methyltransferase involved in the conversion of isoorientin into isoscoparin, however, showed that this activity is always present, independent of the presence of the substrate isoorientin. Further biochemical studies showed that this protein fraction can also catalyze the methylation of caffeic acid to ferulic acid, but not the methylation of cyanidin to peonidine. In order to discriminate between the two possibilities of either a single enzyme with a broad specificity or two different enzymes, the properties of these methylating activities were further investigated. The Km values for the substrates caffeic acid, isoorientin, and isoorientin 2′'-O-rhamnoside were respectively 10, 320 and 7 μM in the presence of saturating concentrations of adenosyl-L-methionine. In the presence of saturating concentrations of the substrates caffeic acid, isoorientin and isoorientin 2′'-O-rhamnoside the Km values for adenosyl-L-methionine were respectively 12, 4 and 5 μM. The Km values at saturating concentrations of isoorientin and isoorientin 2′'-O-rhamnoside were obtained by extrapolation. The V max with caffeic acid is more than three times higher than with isoorientin and isoorientin 2′'-O-rhamnoside, 20, 6 and 3 pmol min -1 mg -1 protein, respectively. With caffeic acid a pH optimum was found at pH7.0, while with isoorientin the pH optimum was at pH8.0. The ferulic acid formation was stimulated by the divalent metal ion Mg 2+ , the isoscoparin formation by the ions Co 2+ and Mn 2+ . Ontogenetic studies on the methyltransferase activities showed that although the caffeic acid and isoorientin methylating activities are present in all stadia of plant development, they are differently regulated. It is concluded therefore that the ferulic acid formation and the isoscoparin formation are catalyzed by different enzymes. Arguments are put forward for the localization of flavone and anthocyanin biosynthesis in different compartments.