Investigations on the formation of dihydrochalcones in apple (Malus sp.) leaves

The apple tree (Malus sp.) accumulates high amounts of the dihydrochalconephloretin and its derivatives. Whereas p-coumaroyl-CoA is the precursor for the naringeninchalcone and further flavonoid formation, p-dihydrocoumaroyl-CoA is required for the biosynthesis of dihydrochalcones. The formation of p-dihydrocoumaroyl-CoA from p-coumaroyl-CoA in apple has been characterized at the enzymatic level whereas the corresponding gene is not unequivocally found. Potential candidate genes including an enoyl-ACP reductase, acyl-CoA dehydrogenase and three different double bond reductases were cloned and heterologously expressed. Activity of the heterologous proteins was tested but none of the genes could be definitely identified as responsible for their involvement in dihydrochalcone formation. In parallel, during a challenging protein purification procedure we isolated for the first time a candidate enzyme from apple leaves, which exhibits strong double bond reductase activity with p-coumaroyl-CoA to form p-dihydrocoumaroyl-CoA. Additionally, aflavonoid 3'-hydroxylase cDNA clone was isolated from young leaves of apple. Heterologous expression in yeast provided functionally active enzyme, but hydroxylation of phloretin was not observed.