School of BioSciences - Theses
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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.