Elucidation of the biochemical pathway leading to the biosynthesis of dihydrochalcone compounds in apple

The apple tree (Malus x domestica) is an agriculturally and economically important tree commonly used in food and beverages. Apple has also drawn attention in recent years due to its potential pharmaceutical and nutraceutical applications which are correlated with secondary metabolites. The major phenolic compounds found in apple belong to the class of dihydrochalcones, represented by various phloretin derivatives (e.g. phloridzin, sieboldin, trilobatin). Phloridzin (phloretin 2’-O-glucoside) also occur beside others in strawberry fruits (Fragaria x ananassa), cranberries (Vaccinium macrocarpon) and sweet tea (Lithocarpus polystachus), but in significant lower amounts compared to apple. Beside their contribution to the bitter taste (flavor) of cider and the colour of apple juices due to oxidation products they were also associated with health effects of apple fruits, berries and their processed products (1). The molecular basis of the biosynthesis of dihydrochalcones has only been partially described so far. Phloretin, the aglycone structure of phloridzin is synthesized via the phenylpropanoid way. By the action of specific or also “unspecific” UPD-glucosyltransferases (UGTs) the regiospecific transfer of a sugar residue from a suitable sugar donor is achieved. Two research groups described putative UGTs involved in phloridzin biosynthesis from apple, whereas Md_PGT1 show high substrate specificity to the chalcone producing exclusively the phloridzin (2). However, recombinant Md_UGT71A15 showed broader substrate acceptance towards other flavonoids and also produces the 4’- and 4-O-glucosides from phloretin in significant amounts (1). Gosch et al. (3) assumed that common chalcone synthases (CHSs) are involved in building the core structure from p-hydroxydihydrocinnamoyl-CoA (Fig. 1), which is proposed to be synthesized by an up to now unknown NADPH-dependent dehydrogenase from p-coumaroyl-CoA. In this study the formation of dihydrochalcone which should involve a double bond reductase activity is under investigation. The exact substrates and the identity of these reductases are still unclear. The extensive apple research and the availability of apple genomic and transcriptomic resources make apple an ideal plant to elucidate this key reductase activity that leads to the production of many valuable dihydrochalcones including potent nutraceuticals and sweeteners. To identify genes involved in the synthesis of dihydro-phenolic compound, like dihydrocoumaroyl-CoA in apple fruits, we screened the existing genome database of the Rosaceae for apple proteins with significant sequence similarity to Arabidopsis alkenal double-bond reductase (AKR). We show here that the functionally expressed apple double bound reductase exhibits coumaroyl-CoA reductase activity generating dihydrocoumaroyl-CoA. Our findings contribute to our understanding of dihydrophenols formation in plants.