School of BioSciences - Theses
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ItemPhylogeography of Box Eucalypts with Disjunct Distributions in South-East Australia( 2021)Despite Australia generally being considered a continent of ancient and stable landscapes, the south-east has experienced major environmental changes during the Pliocene and Pleistocene. In addition to the slow continent-wide decline in rainfall and several global glacial-interglacial cycles, there have been more localised events that have impacted the vegetation of the region including marine inundation of the Murray Basin, uplift of the Padthaway High that dammed the Murray River and formed the large Lake Bungunnia, and volcanic activity in the Newer Volcanics Province of western Victoria and south-eastern South Australia. A record of the impacts of these events on the distribution of plants has been left in the patterns of genetic diversity and relatedness across the landscape. The eucalypts, >850 species of shrubs and trees in the genera Angophora, Corymbia and Eucalyptus, are one of the most widespread and species rich groups of woody plants in Australia, and almost define what is thought of as the ‘bush’. They occur in habitats ranging from wet forests of the east coast and south-west corner of the continent all the way through the climatic gradients to the inland deserts. In my thesis I focus on E. sect. Adnataria, one of the most diverse groups within the eucalypts with ~120 species, and, in south-eastern Australia, the species it contains dominate large areas of open forest and woodlands. This makes members of E. sect. Adnataria good candidates on which to undertake phylogeographic studies to build understanding of the biogeographical history of southeastern Australia, as I have done in this thesis. In chapter 2 I explore the phylogeography of E. behriana using ddRADseq data, showing that the populations of the species west of the lower Murray Basin were isolated earlier than those to the east. This is hypothesised to be related to the marine inundation and formation of Lake Bungunnia in the Murray Basin over the last several million years. The isolation of the populations at Long Forest and around West Wyalong are shown to have recently experienced geneflow with the larger populations of the Victorian Goldfields, Wimmera, and Murray Mallee in the east. This more recent isolations of eastern populations are hypothesised to be due to climate changes under the global glacial cycle, with a possible contribution from recent volcanic activity in the Newer Volcanics Province. I set out to resolve the relationships and taxonomy within the E odorata complex in chapter 3, however I didn’t manage to achieve this using both ddRADseq and DArTseq, with many relationships remaining unresolved, and many questions remaining unanswered. What I show is that most E. viridis populations, E. aenea and E. castrensis form a lineage sister to the remained of the clade, which includes an E. viridis population previous described as E. viridis var. latiuscula from south-eastern Queensland. I also show hybridisation is common within the complex and with taxa outside it, especially the closely related Grey Boxes, which has contributed to the diversity of morphology in the group. Chapter 4 returns to focus on E. behriana, as in this chapter I present a case study on the pitfalls and flaws in using plastid DNA for phylogeography in the absence of substantial outgroup sampling. I show how, by analysing the ingroup only, a geographically sensible and well resolved pattern of population relationships can be established. However, by drastically expanding my sampling to include extensive representation of co-occurring outgroups, I show these patterns become meaningless due to the large cyto-nuclear discrepancy in the eucalypts. A further phylogeographic study is presented in chapter 5, that of E. baueriana. This taxon occurs in coastal regions of south-east Australia, and I show there is a deep divergence between a south-western lineage and a north-eastern one located in eastern Gippsland. While the cause of this disjunction is not clear, we hypothesise it is related to the occurrence of gallery rainforests in the region that prevent E. baueriana form colonising it’s preferred riparian habitat. The fragmentation of the distribution of the two main lineages is hypothesised to be related to glacial-interglacial climatic cycles; in particular, the changes they cause to sea-levels.
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ItemAn Investigation of flavanone O- and C-methylation in Eucalyptus( 2021)Flavonoid compounds are well recognized for their diverse health-promoting properties in humans. Methylation of flavonoids catalysed by plant methyltransferases is an important modification technique that alters the biochemical properties of the compounds, thereby enabling extension of their promising medicinal effects from in vitro to in vivo. Some Eucalyptus trees accumulate high levels of O-and C-methylated flavanones in their leaves. These compounds have potential medicinal and antimicrobial use. Despite the potential use of Eucalyptus species as a commercial source of these flavonoids, little is known about the underlying mechanism of in planta methylation and biosynthesis of these compounds. Through the isolation and characterization of flavanone C-and O- methyltransferases from Eucalyptus, this research aims to provide a detailed insight into the underlying mechanism of different types of in planta methylation and the process by which these compounds are modified. An integrated analysis of transcriptome and metabolome coupled with in silico methods resulted in a cDNA clone (EnOMT1), catalysing the position-specific O-methylation of a single hydroxyl group in the flavanone pinocembrin. The biochemical characterization showed that EnOMT1 is a regiospecific methyltransferase with strict positional specificity to the 7-hydroxyl of flavonoids. The reduction/absence of activity of EnOMT1 with B-ring substituted flavonoids such as luteolin, apigenin, and quercetin demonstrates the pronounced effect B-ring hydroxyl configuration has on the activity of the enzyme. Several Eucalyptus species belonging to subgenus Eucalyptus (monocalypts) show accumulation of different complements of methylated flavonoids. To further understand the genetic basis of this differential accumulation, three homologous genes of EnOMT1 were isolated and characterised from various species. A homologue (ExOMT1) from a species that does not accumulate any methylated flavanones showed 94% identity to EnOMT1; however, the homologue showed no methylation activity with flavanones in vitro. Gene expression analysis using qPCR showed a comparably lower expression of ExOMT1 to that of EnOMT1.Therefore, the observed difference in activity is likely due to the amino acid difference of the EnOMT1 sequence to ExOMT1. Homology modelling along with site-directed mutagenesis of EnOMT1 further identified residues important for activity. The residues -- namely Trp252, Val253, and Asn256 --play critical roles for EnOMT1 activity. SAM binding residues --namely, Trp148, Phe161, Met165, Asn169, Gln194, Phe216, Asp217, Arg218, Asp237, Met238, Phe239, Lyts251, Trp252, Val253, and Trp257 –– were also predicted with a higher degree of confidence. Genetic basis of flavonoid C-methylation is an understudied area and the limited published information on plant CMT sequences makes the research progress difficult. The work presented in chapter 4 provides interesting insights into flavanone C-methylation in Eucalyptus. Extensive flavanone profiling of ten species from subgenus Eucalyptus was conducted to identify a C-methyl flavanone rich species. Two known C-methyl flavanones, cryptostrobin (monomethylated at position C6) and desmethoxymatteucinol (dimethylated at C6 and C8), were identified in E diversifolia in relative abundance. The sequenced transcriptome of E diversifolia was analysed for the identification of secondary metabolite pathway genes putatively involved in C-methylation. Six putative CMT genes were cloned successfully and analysed for activity. Despite our attempts, no in vitro C-methylation was achieved on the flavanone substrates tested. The data presented in chapter 4 provide a solid platform of knowledge for future research on flavanone C-methylation. The results presented in this thesis represent an important step towards obtaining a greater understanding of flavonoid biosynthesis in Eucalyptus and provide clear insights into the reaction mechanism as well as the active site residues of the enzyme.
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ItemNon-volatile secondary metabolites in foliar oil glands of Eucalyptus species( 2020)Plants synthesise a vast range of secondary metabolites that are stored in specialised cells or organs. The presence of sub-dermal glands rich in volatile terpene essential oils is characteristic of the trees of the genus Eucalyptus (Myrtaceae). Recent studies showed that non-volatile compounds (NVCs), particularly monoterpene acid glucose esters (MAGEs), co-occur with volatile components in Eucalyptus foliar oil glands. The principal aim of this thesis is to characterise MAGEs and other non-volatiles localised to Eucalyptus oil glands and to explore their relationships to the co-housed oil components. Glandular extracts from a range of Eucalyptus species belonging to the two major subgenera, Symphyomyrtus and Eucalyptus, were investigated in Chapter 2. Non-volatiles were extracted from enzymatically isolated glands and analysed using high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS). MAGEs were identified based largely on diagnostic mass spectral fragmentation patterns. MAGEs were the dominant NVC in Symphyomyrtus species. In contrast, the sampled subgenus Eucalyptus species lacked MAGEs, but were rich in phenolics. Volatile oil components were also analysed using gas chromatography with flame ionisation detection and mass spectrometry (GC-FID and GC-MS). Monoterpenes and sesquiterpenes were identified and quantified for each species. In addition, cell suspensions of E. polybractea were successfully established from leaf-derived callus as a potential tool to investigate the biosynthesis of MAGEs. Glandular extracts from subgenus Eucalyptus species rich in non-volatiles containing phenolic moieties were further analysed in Chapter 3. A suite of unsubstituted B-ring flavanones was identified as the dominant glandular NVCs. In addition, flavones, flavanone-O-glucosides, flavanone-b-triketone conjugates, triketone heterodimers and chromone-C-glucosides were also identified. Flavanones were quantified and species-specific variations were observed. This chapter also showed that flavanones are exclusively localised to the glands rather present throughout leaf tissues. A positive correlation was observed between some monoterpenes and sesquiterpenes with total flavanones and particularly with pinostrobin. Interestingly, b-triketones were also found in the volatile extracts of glands from E. suberea and E. brevistylis. The presence of glandular b-triketones was further explored in Chapter 4. A major discovery of this thesis was the occurrence of two gland types in E. brevistylis that differ in their colour and importantly, in their metabolic contents. ‘Sesquiterpene glands’, which are translucent-white in appearance, contained sesquiterpene alcohols. ‘Triketone glands’, which are golden-brown in appearance, contained mostly the b-triketone conglomerone and sesquiterpene hydrocarbon caryophyllenes in lower abundance. None of the glands contained the NVCs identified from the other species in this thesis. The results were consistent in trees from a natural population of E. brevistylis and in glasshouse-grown seedlings and saplings. In addition, ‘Triketone glands’ seem to develop earlier than ‘sesquiterpene glands’ in leaf ontogeny. This is the first identification of such metabolic differentiation of embedded glands from any tree species. The work presented in this thesis revealed many novel aspects of oil glands, particularly in relation to their non-volatile and volatile constituents and how they are related to one other. Overall, the findings of this thesis contribute significantly to the knowledge on Eucalyptus foliar oil gland chemistry and biology.