Potential for selection of new peanut genotypes with enhanced polyphenol antioxidant content
The goal of this project was to determine the potential for the breeding of new peanut genotypes with enhanced polyphenol content and antioxidant capacity. The specific objectives of this project were: 1) to estimate the genotype (G), environment (E) and G x E influence on the expression of polyphenols in a Recombinant Inbred Line (RIL) peanut population; 2) to study the molecular basis of polyphenol biosynthesis in peanuts using the shotgun proteomics approach; 3) to screen for new phenolic compounds in a water-soluble peanut extract using LC-MS/MS and propose phenolic biosynthesis pathways, and 4) to investigate the feasibility of developing a simple nanoparticle-based test for the rapid and low-cost phenotyping of antioxidant capacity for rapid selection of antioxidant capacity in breeding and segregating populations. The genotype (G), environment (E), and G x E influence on antioxidant expression investigated in the RIL population showed significant genotypic and environmental effects, but non-significant G x E effects suggesting strong genetic control and moderate heritability. Quantifications of known polyphenols in selected RILs showed increased ferulic acid, p-coumaric acid, salicylic acid, resveratrol, and daidzein with the enzymatic extraction, indicating the importance of matrix-bound polyphenolic compounds and corresponded to respective ORAC assay values. The shotgun proteomics revealed differential protein abundance in the high antioxidant expressing RIL in carbohydrate and protein metabolism, stilbene and flavonoid biosynthesis, anabolic and catabolic pathways. These metabolic changes contributed towards phenylalanine biosynthesis lead to the central phenylpropanoid pathway and subsequent synthesis of identified polyphenols. Enzymes showing significant differential abundance were mapped to describe metabolic changes and biosynthesis pathways related to high antioxidant expression which have potential use as biomarkers for related compounds. In particular, stilbene synthase-like 3 was expressed 4.8 fold greater in the high antioxidant capacity RIL and hence recognised as a valuable target biomarker for stilbenoid compounds. Based on the enzymes identified in polyphenol synthesis in peanuts by the shotgun proteomics, specific polyphenolic compounds were targeted in LC-MS/MS analysis, resulting in the detection of 21 novel compounds not previously identified in Arachis hypogaea. The metabolic and Yan Yee Poon z3160325 9 biosynthesis pathways using a total of 118 polyphenolic compounds detected in this project were assembled and proposed for peanuts. Shape and size transformation of silver nanodisks (AgND) to nanoprisms (AgNPr) and their reductive capacity was exploited to develop a quick colourimetric antioxidant capacity assay that can facilitate rapid selection of superior breeding lines. A linear dose-response between AgND567 transformation and polyphenol was observed for sinapic acid, t-cinnamic acid, caffeic acid, gallic acid, protocatechuic acid, vanillic acid, and syringic acid, also rutin, polydatin and resveratrol at extremely low concentrations (detection range: 1 x 10-3 M – 1 x 10-6 M). A simple soaking method by extraction of skinless kernel in water was examined showed lower correlation of AgND567 response to ORAC values (R2 =0.015, Pearson r = 0.12 and two-tailed P = 0.63), methanolic extracts from defatted peanut kernels, instead showed a significantly improved correlation ( n= 98 against FRAP assay, Pearson R = 0.52, R2 = 0.27, two-tailed P< 0.0001). With further work on the AgND assay-compatible sample preparation, this assay platform shows potential in the development of a simple and rapid nanoparticle-based antioxidant capacity assay for kernel screening in peanut breeding programs.
Polyphenols, Peanut, Antioxidant, Silver nanoparticles, Proteomics
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