THE UNIVERSITY OF NEW SOUTH WALES SCHOOL OF CHEMICAL ENGINEERING FOOD SCIENCE AND TECHNOLOGY Potential for selection of new peanut genotypes with enhanced polyphenol antioxidant content Yan Yee Poon A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Sydney, Australia February 2020 1 Yan Yee Poon z3160325 2 Yan Yee Poon z3160325 3 Yan Yee Poon z3160325 4 Yan Yee Poon z3160325 COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Date ……………………3/02/2020……………………………… AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Date ……………………3/02/2020.……………………………… 5 Yan Yee Poon z3160325 Dedication Dearest Grandma and Grandpa, Please forgive me for the meagre hours I spent with you in your last days, I hope you'll be proud of the work I've done in the past few years. Thank you for your endless encouragement and for always believing in me. We'll meet again soon. Martha, Though you've left us before the journey was over, I'll treasure the time we shared during placement and always hold onto that "trying spirit" you saw in me - we'll stay forever peanut friends. Wallace, Chris, and Mrs Tong, Thank you for the mentorship; your encouragement and teachings will continue to motivate me throughout my passage in research and in life. 6 Yan Yee Poon z3160325 Acknowledgements I'd like to express my gratitude to the Centre for Advanced Technology Food Manufacture and the Australian Research Council for the financial support with the ARC Industrial Transformation Training Centre PhD scholarship, the Peanut Company of Australia (PCA) for peanut samples used in this thesis and UNSW PRSS travel award for the 2017 APRES conference. I am grateful to my supervisor Associate Professor N. Alice Lee for the opportunity to undertake this project and privilege of receiving the scholarship. Thank you, for the generous time exhausted in teaching and correcting me. My co-supervisor Dr Graeme Wright, "The Peanut King", for his kind guidance and nurture during my time on rural visits and remote teaching me. Both their patience and understanding has been tremendous; thank you for not giving up on me. Dr Sridevi Muralidharan, thank you for your gentle friendship and supervision (Chapter 4); the past years you have been like a sister. You were able to sense my unease before anyone else – your kindness and support, the thoughtful calming calls before and after every challenge relieved me. Dr George Lee, thank you for the group cheer up sessions and your supervision (Chapter 6); the crash courses in inorganic chemistry and nanoparticles were much helpful to a biotechnologist. Thank you to Ms Ying Jenny He for her technical assistance on Sections 6.3.1.1 and 6.3.2. Dan O'Connor, my peanut dealer, for fostering me during placement and also in peanut technical support. All PCA staff, for your kind assistance during placement. Thank you very much to Camillo, John and, Ling for the care and support; you are always looking out for our safety, interests, and welfare. Dr Victor Wong for the HPLC and troubleshooting. Dr Dimitrios Zabaras and Ms Mashid Roohanidezfouli (North Ryde, CSIRO) with LC-MS/MS, Dr Qiang Zhu (EMU, MWAC, UNSW) for Technai TEM and Dr Martin Bucknall (BMSF, MWAC, UNSW) for GCMS. To Dr Maria Chandra-Hioe and Dr Chatchaporn Uraipong for their technical assistance and support. 714 comrades; especially Steffi, Ji, Jun, Yiqing, Scott, Dat, Kornelia, and Johanna: Though we battled our own demons individually, we fought together, shared pain and encouragement; I would not have endured these years alone. Thank you to colleagues at AB Mauri and friends who gave me the mental strength, consolation, and distractions to keep me sane and grounded. Honeypot for the comfort and play-times, you've given much calm, confidence, and relief in times of panic and anxiety. Finally, but most importantly, I want to thank my parents and my sister for their unwavering understanding, assurance, and their faith in my choice of path, though it inconvenienced them greatly, never protested. The unconditional love, optimism, and reassurance shown through the pressures of life, sickness, and death - I am thankful for. 7 Yan Yee Poon z3160325 Abstract 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 8 Yan Yee Poon z3160325 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 2 response to ORAC values (R =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. 9 Yan Yee Poon z3160325 List of Abbreviations ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) AEA Average environment axis AEC Average environment coordination AFLP Amplified fragment length polymorphism Ag+ Silver ion AgNP Silver nanoparticle ANOVA Analysis of variance AU Absorbance unit AuNP Gold nanoparticle BSA Bovine serum albumin BSPP Bis(ρ-sulfonatophenyl) phenylphosphinedihydrate dipotassium salt solution BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide CCV Continuing calibration verification CHD