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dc.contributor.advisorPerry, Nigel
dc.contributor.advisorLarsen, David
dc.contributor.authorRichardson, Alistair Thomas Bloor
dc.identifier.citationRichardson, A. T. B. (2020). Chemistry of a Stable Vitamin C Glycoside in Apples (Malus spp.) and Other Fruit Crops (Thesis, Doctor of Philosophy). University of Otago. Retrieved from
dc.description.abstractThe primary aim of the research described in this thesis was to investigate an ascorbyl glycoside tentatively identified in crab apples (Malus sylvestris) by a non-targeted metabolomics study (unpublished thesis, J. Cho, 2016). The interest in this metabolite arose from the possibility that it may be involved in the translocation of ascorbate from leaf to fruit in apples. This hypothesis was based on its similarities to tutin-glycosides and sucrose-glycosides which are utilised for long distance translocation in Coriaria and Cucurbita spp. respectively. The chemotaxonomy of the Coriaria genus was also investigated as part of this research (Appendix 2). Isolation of the target ascorbyl glycoside from crab apples was achieved via acetylation of the crude extract and subsequent purification. This compound was definitively characterised by comparison to a synthetic standard and found to be 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA2βGlc). This derivative was found to have greater stability than ascorbate and was comparable to AA2αGlc, which is commonly used in cosmetic products as stabilised pro-vitamin C. This was only the third reported occurrence of an ascorbyl glycoside in plants, and the second report of this specific metabolite. Quantitative HILIC LC-MS was used to analyse leaf and fruit tissue of five apple genotypes at three separate collection dates representing the start, middle and end of fruit development. Crab apples were found to have an extremely high concentration of AA2βGlc in their fruit (>500 mg/100 g FW), but only trace quantities (<10 mg/100 g FW) were detected in fruit of domestic cultivars (Braeburn and Royal Gala). Leaves of all varieties contained AA2βGlc at similar concentrations (approx. 30 mg/100 g FW). A significant increase in AA2βGlc concentration was observed between the start and middle of fruit development (October-December). Collection and analysis of fruit and leaf material from an additional 12 fruit crops, predominantly of the Rosaceae family, revealed that they also contained AA2βGlc in their leaves. While previously thought to be rare, these results extend the known occurrence of this metabolite to 14 species and five families. Based on the distribution in leaves and fruit it was proposed that AA2βGlc was utilised as a stabilised form of ascorbate for translocation between leaves and fruit. In order to provide evidence for this hypothesis phloem exudates of five apple genotypes were analysed by HILIC LC-MS. AA2βGlc was present in phloem of all genotypes, but ascorbate was found at greater concentrations, which showed that AA2βGlc was not the major form of translocated ascorbate. Glucosidase assays were used to determine if enzyme activity indicative of phloem unloading, i.e. hydrolysis of AA2βGlc to ascorbate, was present in any of the five apple genotypes. Protein extracts of stems and leaves were found to have generic β-glucosidase activity, but none of the tissue types tested showed activity towards AA2βGlc. Further work is required to understand the biological role of this metabolite in apples. The synthesis of four ascorbyl glycosides was achieved, establishing the structure of the naturally occurring metabolite and providing a valuable resource to the experiments described above. Previously unreported routes to access AA2βGlc and AA2βGal, as well as methodology towards AA5βGlc and AA6βGlc, are described. Inconsistencies in the reported NMR data for ascorbyl glycosides were clarified by demonstrating that these spectra were highly pH dependent. This work dramatically enhances what is known regarding ascorbyl glycosides in plants and provides a foundation from which to determine the physiological role of this previously overlooked metabolite. Further work should focus on more rigorous biological experiments to determine whether the accumulation of AA2βGlc in crab apples is a result of translocation from leaves or in situ biosynthesis. This may lead to the development of cultivars with increased vitamin C concentrations, consequently adding both nutritional and commercial value to one of New Zealand’s largest horticultural exports.
dc.publisherUniversity of Otago
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dc.subjectVitamin C
dc.subjectAscorbic acid
dc.subjectascorbyl glycoside
dc.subjectcrab apple
dc.titleChemistry of a Stable Vitamin C Glycoside in Apples (Malus spp.) and Other Fruit Crops
dc.language.rfc3066en of Philosophy of Otago
otago.openaccessAbstract Only
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