Epidemiological studies suggest positive health benefits from a diet rich in fruits and vegetables. This is often attributed to the abundant phytochemical content. The health-promoting effects of phytochemicals are one of the essential properties of fruits and vegetables, however the accumulation of phytochemicals in fruits and vegetables can vary amongst different plant species and species varieties.
Efforts to study and improve phytochemical content have been carried out on various temperate fruits. In contrast, little research has been conducted on tropical fruits. Mango (Mangifera indica L.) is one of the significant important fruit crops in the world, and mango fruits are a potential source of bioactive compounds. There are hundreds of mango varieties whose fruits show a pronounced diversity in size, shape, colour and flavour. Mango is a good candidate for research on phytochemical improvement through breeding and/or selection to create better fruit value, because of the wide genetic diversity and the current lack of breeding for nutritional quality. From the studies presented in this thesis, possible links between mango genes and biological properties of phytochemicals identified in ripe mango fruits were assessed. Three genetically diverse commercial mango varieties Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) were chosen for investigation.
The first part of the study compared the bioactivities of extracts prepared from the peel and flesh of the three mango varieties. The total polyphenolic content of extracts and preparative HPLC fractions was measured by the Folin-Ciocalteu reagents and associated biological activities were assessed in breast cancer cell-based assays. NDM represented a mango variety with particularly high levels of fruit peel bioactivity, as assessed by the ability to reduce viable cell number and induce cell death in human breast cancer cell lines. We then compared the presence of major polyphenolics in three extract and the amounts of flavonoids, phenolic acids, and hydrolysable tannins to understand their proportion in each mango extracts and their possible relationship with bioactivity. Despite the major differences in bioactivity only subtle differences in variety-specific phytochemicals were found. This suggests that the particular combination of polyphenols may be responsible for the greater bioactivities of NDM peel extracts.
The phenylpropanoid-flavonoid pathway is responsible for the biosynthesis of the major bioactive compounds identified in the extracts of all mango varieties. This pathway has been extensively investigated in a wide range of plant species due to the putative nutraceutical benefits of the secondary metabolites generated by this pathway. However, this pathway has not yet been investigated in mangoes and other tropical fruits. To understand the phenylpropanoid-flavonoid pathway in mango I proposed a pathway constructed based on a combination of automated pathway data extraction from Kyoto Encyclopaedia of Genes and Genomes database (KEGG) and the presence of selected flavonoids in mango fruit extracts. In this study, expressed sequence tags (ESTs) were used to identify putative genes of the phenylpropanoid-flavonoid pathway of mango. I identified for the first time, full or partial sequences corresponding to ten out of twelve predicted mango flavonoid biosynthesis enzymes. A close evolutionary relationship between mango genes and those from the woody species such as poplar (Populus trichocarpa) and grape (Vitis vinifera), was revealed through phylogenetic analysis. Bioinformatics analysis showed a high degree of similarity in their deduced amino acid coding sequences and indicated that the phenylpropanoid-flavonoid pathway proteins in mango and other dicotyledonous plants are highly conserved.
Because nucleotide sequences contained in the mango ESTs database were derived mostly from complementary DNA libraries of KP and IW leaf tissue, sets of universal mango primers were designed and successfully used to amplify the biosynthetic genes of KP, IW and NDM varieties. All PCR products were cloned into plasmid vectors for sequencing and genetic polymorphisms between mango varieties were examined. Although numerous single nucleotide polymorphisms (SNPs) were detected, they did not appear to lead to significant amino acid changes, such as premature stop codons in the predicted protein sequence. In most cases, sequence comparison analyses revealed that the phenylpropanoid-flavonoid proteins showed a high similarity between the three different mango varieties tested, indicating a possible conservation of their biological roles within these groups. I also discovered an association between sequence variation and position in the pathway for up-stream genes. The nucleotide and non-synonymous variation was found to be low in phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS).
The final part of this research addressed the association of the identified mango fruit phytochemicals and the differential expression of their biosynthetic genes. Gene expression measured by quantitative real-time RT-PCR displayed differential expression profiles from the different parts of mango fruits. In every examined mango variety, mango peel exhibited higher transcript levels than flesh for PAL, C4H and CHS genes, which are thought to control flux into the flavonoid pathway. Results suggest that PAL, C4H and CHS enzyme expression have an influence on total flavonoid levels in mango fruits. In addition, the particularly high expression levels of anthocyanin reductase (ANR) in KP and NDM peels was coincident with epicatechin digallate (ECG) accumulation in those extracts. This result suggests that ECG biosynthesis is likely controlled by ANR in mangoes. The transcription of 4-coumarate:CoA ligase (4CL) gene tended to be higher in KP peel than IW and NDM peels, which supports the role of 4CL on mangiferin accumulation in KP peel.
Collectively, my studies provide further evidence of the significant bioactives diversity between genetically diverse mango fruit varieties. Based on the EST re-sequencing, I identified ten genes that direct mango flavonoid biosynthesis. This thesis also reports an extensive comparison of these genes in three genetically diverse mango varieties. It extends current knowledge about the structural and functional characteristics of the biosynthetic genes in this pathway. This work also establishes a relationship between chemically identified flavonoid compounds in mango fruit extracts and transcriptional profiles of the underlying phenylpropanoid-flavonoid pathway genes in mango fruits. This is important because it will assist the future genetic manipulation of mango to produce new varieties with a higher concentration of bioactive compounds of benefit to human health.