Chalcone synthesis with enzyme extracts from tulip anther tapetum using a biphasic enzyme assay

The in vitro synthesis of chalcones has been demonstrated using a special biphasic enzyme assay. The highly viscous lower phase in this assay stems from a tapetum fraction of anthers of Tulipa cv. “Apeldoorn” which has been used an enzyme source. The upper phase of this system consists of a reaction mixture of the normal “flavanone synthase” assay. It is suggested that chalcone synthesis occurs at the boundary layer between the two phases. To prevent spontaneous as well as enzymatic cyclization of the chalcones formed (phloroglucinyl type), the pH of the upper phase must not be allowed to exceed pH 4.0. Under these pH conditions, chalcone formation by a reverse reaction of chalcone-flavanone isomerase can be excluded. The measured substrate specificity of the “chalcone synthase” corresponds to the conditions of chalcone formation in the natural system. Using p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, respectively, as substrates, the enzyme system forms the correspondingly substituted chalcones which are also accumulated in the loculus of tulip anthers. It is suggested that this chalcone synthase is identical to the previously described “flavanone synthase”. The results can be further explained as follows. (i) Not flavanones, but rather chalcones are the first C₁₅ intermediates of flavonoid biosynthesis in tulip anthers. (ii) In this Tulipa system, the substitution pattern of three different hydroxycinnamic acids can be transferred unchanged into the flavonoid C₁₅ stage. (iii) The role of chalcone-flavanone isomerase is to cyclize chalcones to flavanones on the direct biosynthetic pathway to the further accumulated flavonol glycosides. (iv) The sensitivity of the reaction with regard to chalcone production points to the localization of chalcone synthase in a most unstable and, up to now, unknown tapetal compartment. Since purification of the enzyme results in exclusive production of flavanones, it is suggested that certain “chalcone stabilizing factors” must occur in the natural system. (v) The phenomenon of chalcone accumulation in tulip anthers, however, must be caused by a complex system, distinguished by cooperation of certain biochemical and physiological conditions, and, finally, by special compartmentation of the enzymes which are responsible for the biosynthesis of flavonoids.