Understanding the Biosynthesis of Phloridzin in Apple

Abstract

The dihydrochalcone phloridzin is the major phenolic compound found in apple (Malus x domestica) and is present in high concentrations in the bark, roots and leaves. Dihydrochalcones have been been found in very small quantities in other genera but high concentrations of phloridzin are only found in apple. The phloridzin pathway is a branch of the phenylpropanoid pathway and an unknown carbon double bond reductase (CDBR) is believed to be the key branch point enzyme. The primary aim of this thesis was to identify the CDBR enzyme and to gain an understanding of why high concentrations of this dihydrochalcone are unique to apple. Candidate CDBR genes were identified by reference to previously characterised reductases acting on structurally similar substrates. Three classes were identified; alkenal reductases, isoflavone reductases and enoyl CoA reductases. Apple genes sharing sequence similarity to these CDBRs were identified from published ESTs and tested using two over expression systems (stable expression in tt5 Arabidopsis and transient expression in 35S:AtMYB75 tobacco). An RNAi silencing strategy in ‘Royal Gala’ was adopted to test the effects of candidate gene suppression on phloridzin levels. Finally, expression levels of phenylpropanoid genes in the leaves of apple and pear were compared to identify potential expression patterns which might lead to phloridzin production in apple. A total of 13 candidate genes were tested for their role in phloridzin biosynthesis using the strategies described above. One enoyl CoA reductase enzyme (ENRL-3) was able to increase the levels of phloridzin when transiently expressed in 35S:AtMYB75 tobacco leaves. Silencing ENRL-3 in ‘Royal Gala’ led to a 67% decrease in phloridzin levels in the one available silenced line. In addition, an ENRL enzyme closely related to ENRL-3 reduced the putative phloridzin pathway substrate p-coumaroyl CoA. Expression studies and enzyme assays revealed lower CHI (CHALCONE ISOMERASE) transcript and activity levels in apple compared with pear. Futhermore, enzyme assays using ‘Royal Gala’ protein extracts showed the reduction of naringenin chalcone to phloretin, implying the existence of a novel phloridzin pathway branch point at naringenin chalcone. Collectively these results suggest that ENRL-3 contributes to the total pool of phloridzin in apple leaves. However, there may be other enzyme activities which led to phloridzin formation in apple. The low CHI expression observed in apple leaves and the ability to form phloretin from naringenin chalcone, instead suggest that a unique metabolite bottleneck exists at this step which results in the formation of high levels of phloridzin seen in apple. This study provides the platform for future studies into phloridzin biosynthesis and in particular elucidation of the newly observed naringenin chalcone reductase activity.