THE CHEMICAL and GENETIC BASIS of TOMATO FLAVOUR Piotr
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THE CHEMICAL AND GENETIC BASIS OF TOMATO FLAVOUR Piotr Jasionowicz, MSc. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy July 2012 ABSTRACT Tomatoes often lack the characteristic good taste that consumers expect. In recent years, the fruit and vegetable industry has focused on traits such as yield, colour, and fruit size; however, flavour has been largely forgotten, at least for varieties that are grown in bulk and are cheap to purchase. Tomato volatiles are one of the most important flavour contributors along with sugars and organic acids. Over 400 volatiles are present in tomato, but only about 30 are considered critical to tomato flavour. The purpose of this study was to identify volatile Quantitative Trait Loci (vQTL) using publicly available Solanum pennellii and Solanum habrochaites introgression lines (ILs). Detection of vQTL is an essential milestone for identification of candidate genes involved in tomato flavour. Identification of vQTL was undertaken by screening the ILs using Atmospheric Pressure Chemical Ionisation Mass Spectrometry (APCI-MS) and Gas Chromatography Mass Spectrometry (GC-MS). Additionally key volatiles and their interactions were evaluated in a series of sensory experiments using tomato juice and tomato purée. The study revealed that, the S. pennellii population had several major effects including those on IL1-4, which had a vQTL for C6 volatile E-2-hexenal. It was possible to identify a sub-IL, 1-4-1 that also harboured this vQTL. In this IL1-4- 1 region of chromosome 1, 122 genes were present including a possible, candidate gene likely to be involved in lipoxygenase pathway. The sensory experiments showed strong impact of methyl salicylate on other volatiles; E-2- hexenal and citral showed positive effects in purée studies. Determined taste detection thresholds for volatiles in tomato juice and purée were established. Keywords: volatile, genes, tomato juice, tomato purée, flavour i “It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity, it was the season of Light, it was the season of Darkness, it was the spring of hope, it was the winter of despair, we had everything before us, we had nothing before us, we were all going direct to Heaven, we were all going direct the other way . ." Charles Dickens “A Tale of Two Cities” I dedicate this thesis to my parents ii ACKNOWLEDGEMENTS I would like to acknowledge the other researchers who dedicated their lives to science, and to their work, that greatly influenced and extended our current knowledge in the field of Plant Science, especially the tomato. Big thank you I would like to say to Graham Seymour for his continuous advice during the PhD programme, and for being an excellent mentor. I also appreciate and I am grateful for the advice from Andy Taylor and Rob Linforth in Food Sciences for helping me understand the complexity of flavour, especially with my initial little understanding of food and flavour chemistry. I acknowledge Charles Baxter from Syngenta for his guidance, Louise Hewson and Joanne Hort in Sensory Science Centre for help with the design of sensory experiments. I would like to thank Syngenta and BBSRC for kindly providing me the financial support. I would like to acknowledge my colleagues and friends in Graham Seymour group, who have helped me greatly during this work, namely Mervin Poole, Matthew Jones, Natalie Chapman, Viv Taylor, Rebecca Smith, Nathalie Naraidoo, Yu Pan, Subhalai Jayasuta, Fereshteh Malekpoor and Guiping Sun. I would also like to thank Bee Lynn Chew and Kunal Saini for some good advice and support during this PhD. You two are good friends. I also wish to thank other people whose support and knowledge significantly contributed to this study: Neil Graham for help with GeneSpring, Wassia Dussatoir for big help and support with tomato juice experiments, Quin Jiang for assistance with tomato purée experiments, Dani Zamir and Uri Krieger for kindly providing tomato seeds for research. Special thanks go to Avinash Kant for help with APCI-MS and GC-MS and to Rob Linforth for teaching me about iii instrumental analysis. Work with some of you, contributed highly to the progress of the study presented in this thesis. Special thanks go to all of the people from School of Biosciences not mentioned here: School office staff, technical staff, and other lab colleagues. Special thanks go to my parents for their guidance and patience throughout my whole life. Finally, I would like to thank all my friends for being always there for me. I hope wherever we will go, we will never be forgotten. iv Table of Contents ABSTRACT ...................................................................................................... i ACKNOWLEDGEMENTS ............................................................................... iii Abbreviations ................................................................................................ viii List of Tables .................................................................................................. xi List of Figures ............................................................................................... xiii CHAPTER 1: .................................................................................................. 1 Introduction and literature review .................................................................... 1 1.1 Tomato History ...................................................................................... 1 1.2 Economic importance and nutritional value of tomato ............................ 3 1.3 Tomato biology ...................................................................................... 6 1.4 Morphology of the tomato fruit ............................................................... 9 1.5 Fruit development and ripening. ...........................................................10 1.5.1 The Tomato cell wall ......................................................................16 1.5.2 Fruit colour .....................................................................................21 1.5.3 Generation of flavour .....................................................................24 1.5.4 Taste perception ............................................................................25 1.5.5 Tomato taste ..................................................................................28 1.5.6 Aroma ............................................................................................28 1.6 Metabolomics .......................................................................................48 1.6.1 Metabolomics overview ..................................................................48 1.6.2 Technology for metabolomics analysis ..........................................50 1.6.3. Metabolomics and the tomato .......................................................51 1.6.4 Experimental design for chemical and genetic basis of tomato flavour ....................................................................................................52 1.7 Strategies for crop improvement...........................................................63 1.7.1 Genetically modified tomatoes .......................................................63 1.7.2 Targeting Induced Local Lesions in Genomes (TILLING) ...............65 1.7.3 Harvesting natural variation ...........................................................68 1.8 Significance of this work .......................................................................73 AIMS OF THE PROJECT ..............................................................................76 CHAPTER 2: .................................................................................................77 High throughput screening of tomato volatiles by Atmospheric Pressure Chemical Ionisation Mass Spectrometry (APCI-MS) ......................................77 v 2.1 Introduction ..........................................................................................77 2.1.1 Maceration of fruits to mimic volatile release on eating ..................77 2.2 Materials and Methods .........................................................................78 2.2.1 Plant material .................................................................................78 2.2.2 Growth conditions ..........................................................................79 2.2.3 Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) ..............................................................................................80 2.2.4 Normalisation procedure ................................................................85 2.2.5 Statistical analysis .........................................................................85 2.3 Results and Discussion ........................................................................86 2.3.1 Overview of volatile metabolites detected by APCI-MS: results from the S. pennellii ILs ..................................................................................86 2.3.2 Detailed analysis of the distribution of key volatiles as determined by APCI-MS ................................................................................................97 2.3.3 Overview of volatile metabolites detected by APCI-MS: results in the S. habrochaites ILs ............................................................................... 111 2.3.4 Detailed analysis of the