Characterisation of Flavonoid Composition and Glycosylating Enzymes in Sweet Cherry and Apple Fruit
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Date
2023
Authors
Clayton-Cuch, Daniel James
Editors
Advisors
Collins, Helen
Bulone, Vincent (Flinders University)
Bottcher, Christine (CSIRO)
Bradley, David (Agilent Technologies)
Bulone, Vincent (Flinders University)
Bottcher, Christine (CSIRO)
Bradley, David (Agilent Technologies)
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Thesis
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Abstract
Polyphenolics represent a prominent group of secondary metabolites found in fruits and vegetables, with the potential to confer various health benefits to humans upon ingestion, primarily attributed to their antioxidant and anti-inflammatory properties. Among polyphenols, flavonoids, the most abundant subset of polyphenolic compounds, have garnered significant scientific attention in recent years, owing to their structural diversity and various health-promoting properties. An increase in the demand for functional foods, particularly those rich in these beneficial compounds, has served as the primary catalyst for the research in this area. This interest has popularized techniques aimed at enhancing flavonoid content in fruits and vegetables. However, to enhance flavonoid content in these plants, it is important to first characterize the biosynthetic pathways and associated genes to identify optimal candidates for precisely controlling the concentrations of these health-promoting compounds. Glycosyltransferases (GTs), specifically UDP: flavonoid-glycosyltransferases (UFGTs), are responsible for the glycosylation of flavonoid aglycones, which transfer a sugar that is linked to uridine disphosphate (UDP) to these metabolites through an inverting catalytic mechanism. Glycosylation of flavonoids increases the diversity of the properties of these compounds, providing structural stability and higher solubility, for example. All GTs responsible for catalysing flavonoid conjugation belong to the GT1 family, which is the largest characterized group of GTs of the total 116 families. Improving the understanding of GTs will inform researchers on the choice of gene for manipulation of plant systems or expression in heterologous systems for large-scale production of flavonoids. Initially, this thesis presents a comprehensive literature review of the structure, abundance, biological activities, biosynthesis and exploitation of flavonoids in Chapter 1. In Chapter 2 of this work the biosynthesis of an important sub-class of flavonoids, anthocyanins, in the cherry fruit (Prunus avium L.) was improved through the application of the exogenous synthetic auxin 1-napthaleneacetic acid (NAA) hormone at a specific phase of fruit ripening. NAA-treated fruits exhibited higher concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the primary storage form of abscisic acid (ABA), ABA-glucosyl ester (ABA-GE). These changes in hormone levels were associated with changes in anthocyanin biosynthetic genes, as well as the concentrations of anthocyanins found in the fruit. These data suggest that NAA treatment alters ethylene production, which in turn induces ripening in sweet cherry and enhances anthocyanin production, possibly through ABA metabolism. Later, the key glycosylation step in the flavonoid biosynthesis pathway of both apple (Malus domestica L.) and sweet cherry was investigated through the identification of UFGT genes and characterisation of the associated proteins. The glycosylation step is of significant interest due to the capacity of this modification to improve the stability and solubility of flavonoids, therefore improving bioavailability associated with consumption. In Chapter 3, 234 putative glycosyltransferases involved in apple flavonoid biosynthesis were identified, from which the biochemical and structural characterisation of MdUGT78T2 was demonstrated. This enzyme was found to be a strict galactosyltransferase primarily involved in the formation of quercetin-3-O-galactoside and cyanidin-3- O-galactoside, the major glycoconjugates of flavonoids in apple. This newly discovered type of catalytic activity can potentially be exploited for in vitro modification of flavonoids to increase their stability in food products and to modify apple fruits or other commercial crops through breeding approaches, ultimately enhancing their health benefits. Further in Chapter 4, we reported on the biochemical characterisation of two more such enzymes in Prunus avium L., which are poorly characterized particularly in this species. The two glycosyltransferases were named PaUGT1 and PaUGT2. Both enzymes were found to be promiscuous glucosyltransferases active on diverse anthocyanidins and flavonols, as well as phenolic acids in the case of PaUGT1. The expression of the gene encoding PaUGT1, the most active of the two proteins, follows anthocyanidin accumulation during fruit ripening, suggesting that this enzyme is the primary glycosyltransferase involved in flavonoid glycosylation in sweet cherry. These enzymes can potentially be used to synthesize diverse glycoconjugates of flavonoids for integration into bioactive formulations, and for generating new fruit varieties with enhanced health-promoting properties using diverse breeding methods. The output of this thesis has advanced our understanding of key enzymes in apple and cherry responsible for the glycoconjugation of flavonoids as well as the hormonal regulators of anthocyanin biosynthesis, which are findings of both scientific and commercial interest. Finally, the portion of the industry placement associated with this industry PhD program describes the development of a method using ICP-OES 5800 instrumentation for the precise identification and quantification of titanium dioxide (TiO2) from a range of food products (Appendix 1). TiO2 has been used in a diverse range of industries since its initial discovery and is added to foods primarily as a colouring (whitening) agent. Following the recent ban of TiO2 in all food products within the European Union, this chapter addressed a significant issue that many food manufacturers are facing considering these recent bans, by developing a simple, quick, and robust method for the analysis of this food additive within various food products.
School/Discipline
School of Agriculture, Food and Wine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2024
Provenance
This thesis is currently under embargo and not available.