Understanding the Biosynthesis of Phloridzin in Apple

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dc.contributor.advisor Greenwood, D en
dc.contributor.advisor Hellens, R en
dc.contributor.author Dare, Andrew en
dc.date.accessioned 2013-08-23T04:10:57Z en
dc.date.issued 2013 en
dc.identifier.uri http://hdl.handle.net/2292/20710 en
dc.description.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. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA99245308714002091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/3.0/nz/ en
dc.title Understanding the Biosynthesis of Phloridzin in Apple en
dc.type Thesis en
thesis.degree.discipline Biological Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The Author en
dc.rights.accessrights http://purl.org/eprint/accessRights/OpenAccess en
pubs.elements-id 405867 en
pubs.record-created-at-source-date 2013-08-23 en


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