Chalcone 3-hydroxylase

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. Differences in the flavonoid metabolism of transgenic Arabidopsis lines will show the influence of CH3H on the hydroxylation pattern of chalcones, aurones and other flavonoid classes. Sequence comparison of CH3H/F3`H pairs from different classes will indicate candidate sites determining chalcone acceptance which will be confirmed by structure function relationship studies. This will lay the foundation for understanding the amino acid compositions of the active site cavities, the reaction mechanisms catalyzed, the substrate specificities, and the phylogenetic relationships. The CH3H promoter will be studied in transgenic tobacco plants harbouring chimeric ch3h promoter::GUS constructs.

The project studied pigment formation in plants and fed into 12 publications (8 accepted, 1 submitted, 3 in preparation), 8 conference contributions, 3 master thesis and 1 PhD thesis. The results also initiated an EU-project which will train 6 PhD students for four years in breeding of innovative novelties within the European Industrial Doctorates project FlowerPower (675657 FLOWERPOWER-H2020-MSCA-ITN-2015.) Coloration is an essential asset for the plants survival (e.g. signals, UV protection, light harvest) and also an important determinant of consumer preference and marketability of flowers and plant derived food, in the latter case not only for optical effects, but also for the health beneficial value that is associated particularly with red and yellow pigments. The project focused on chalcone 3-hydroxylase (CH3H), a key enzyme in the formation of special yellow pigments, the so-called anthochlors. CH3H was discovered recently in the ornamental plant Cosmos sulfureus and shows a specialized substrate specificity in comparison to the closely related flavonoid 3-hydroxylase (F3H) which is involved in the formation of the important bioactive flavonoids. It is likely that CH3H developed from F3H as an adaptation to pollinators who are attracted by yellow coloration. The project identified novel CH3H and F3H genes from horticultural plants. The encoded proteins were characterized in detail. New insights in their substrate specificity were obtained. Sequence comparison of F3H and CH3H and production of mutants with specifically exchanged amino acids identified regions determining the substrate specificity. We were able to convert an F3H into a CH3H and vice versa. It became, however, obvious that the chalcone acceptance exclusively found for CH3H can be influenced by slight changes in the active center. For a full understanding of the substrate specificity data in correlation with the amino acid sequence, the availability of the CH3H crystal structure will be essential. As a preparation for this, different systems and purification tags were tested for the large-scale production of recombinant CH3H. Application of the optimized system in a follow up project is foreseen. In addition we showed for the first time that in many plants F3H and CH3H can act at an unexpectedly late stage of the flavonoid biosynthesis by accepting leucoanthocyanidins as substrate. Strawberry F3H, however, is not able to do so and this is an essential precondition for the accumulation of the typical bright red pigments of strawberry fruits. F3H and CH3H were also shown to accept the rare dihydrochalcones as substrates and to initiate the accumulation of the so-called 3-hydroxyphloridzin in apple. Genetically modified apple trees harboring the CH3H turned out to be less susceptible to apple scab and fire blight, the most important apple diseases. The physiological relevance of CH3H for aurone formation in Asteraceae species by providing the preferred precursors for the PPO that catalyzes aurone formation in the subsequent biosynthetic step was demonstrated.