This proposal focuses on open questions of chalcone hydroxylation. This plays an important role in the biosynthesis of anthochlor pigments (chalcones and aurones), which provides yellow flower colour in a number of ornamental plants, form UV-honey guides in certain Asteraceae species and also show health-beneficial effects of chalcones in humans. Furthermore, chalcones are the immediate precursors for the formation of flavonoids, which are important compounds in plant-derived human diets and of isoflavonoid-based phytoalexins. The cytochrome P450 dependent CH3H catalyzes the introduction of a hydroxyl group in position 3 of chalcones. The reaction shows high similarity to the hydroxylation of flavonoids in position 3’ but cannot be catalyzed by the prominent flavonoid 3’-hydroxylase (F3’H) despite its broad substrate specificity. A cDNA clone encoding an enzyme with specific CH3H activity was detected only recently by the applicant in yellow flower petals of Cosmos sulphureus (Accession No FJ216429). Compared to its high CH3H activity, the enzyme showed a low F3’H side activity in vitro indicating that CH3H has probably evolved from F3’Hs. The substrate specificity in planta was not investigated so far. Apart from substrate acceptance, the specific expression of ch3h in flowers and germinating seedlings is the second striking difference of CH3H compared to the ubiquitously expressed F3’H. The goal of the present project is (i) to identify for the first time the molecular base of CH3H for chalcone acceptance, (ii) to investigate substrate specificity and the contribution of CH3H to aurone formation in planta and (iii) to study the CH3H promoter and the tissue specific expression.