“Polyphenolic and Proteomic Characterization of Malus domestica Fruit Affected by Bitter Pit” TESIS DOCTORAL Michael Owen Krawitzky Directed by: Dr. Jesus Val Falcon Dra. Rosa Oria Almudí Universidad de Zaragoza Facultad de Veterinaria Dpto. Producción Animal y Ciencia de los Alimentos Zaragoza, 2015 2 Los directores de la Tesis Doctoral de Michael Owen Krawitzky que, lleva por título “Polyphenolic and Proteomic Characterization of Malus domestica Fruit Affected by Bitter Pit”, el Dr. JESUS VAL FALCON, director de la Estación Experimental Aula Dei (EEAD) - Consejo Superior de Investigaciones Científicas (CSIC) y la Dra. ROSA ORIA ALMUDÍ, Catedrática de Tecnología de los Alimentos de la Facultad de Veterinaria de la Universidad de Zaragoza y CERTIFICAN: Que el doctorando realizó una estancia de investigación en el área de “Microbiologie de l’alimentation au service de la santé (MICALIS)” del Institut National de la Recherche Agronomique (INRA), en el centro de Jouy-en-Josas (Francia) entre el 2 de Julio y el 19 de Diciembre de 2012. Durante su estancia, el doctorando desarrolló una serie de ensayos experimentles que forman parte de su Tesis Doctoral y se formó en el empleo de distintas técnicas analíticas avanzadas. Su estancia se realizó bajo la supervisión de la Dra. Veronique Monnet, responsable del grupo “Proteomic Analysis Platform of Southwest Paris” - (PAPPSO). La Dra. Monnet obtuvo su título de Doctor en la Universidad de Paris Sud-Sciences (ORSAY) (anteriormente “Université d’Orsay”) con fecha de expedición del 11 de Julio de 1986. Los informes favorables de la memoria de dicha Tesis Doctoral han sido realizados por la Dra. Jessica Torrion, investigadora y profesora asociada de la Universidad de Montana (EEUU) que es Doctora por la Universidad de Texas Tech. (EEUU) con fecha de expedición del título de 3 de diciembre de 2008, y por la Dra. Laura Soler, investigadora en INRA (Centre Val de la Loire, Francia) y Doctora por la Universidad de Murcia con fecha de expedición del título de 16 de Junio de 2010. Y para que así conste a los efectos oportunos, firma la presente en Zaragoza, a veinticuatro de Abril de dos mil quince. Fdo. Jesus Val Falcon Fdo. Rosa Oria Almudí 3 4 This study was supported by the projects AGL2009-59 08501 (Programa Nacional de Proyectos de Investigación Fundamental), co-financed by the European Social Fund and by the Departamento de Ciencia, Tecnología y Universidad del Gobierno de Aragón, Research Group “Foods from Plant Origin”. Fellowship funding JAE- Predoc/CSIC was supported by the Ministerio de Economía y Competitividad (MINECO). 5 6 Preface The present doctoral thesis has been developed on the frame of the Doctorate Program “Quality, Safety and Technology of Food” from the Faculty of Veterinary of University of Zaragoza. The thesis has been developed on the Plant Food Research Group, which is an interdisciplinary group formed by researchers of four institutions from Aragon: University of Zaragoza (Unizar), Spanish National Research Council (CSIC), Aula Dei Scientific and Technological Park (PCTAD) and Agrifood Research and Technology Centre of Aragon (CITA). The experimental work has been mostly performed in the Plant Nutrition Department of the Aula Dei Experimental Extension (CSIC), whose main research line is the pre- harvest factors affecting fruit quality, in particular those related to calcium intake. This thesis is presented in the modality of “International Doctorate”, according to the Article 18, Title IV, Chapter II from the Agreement of the 20th of December of 2013 approved by the Government Council of University of Zaragoza, containing the Doctoral Thesis Regulations (BOUZ 10/01/2014). To qualify for this international mention, Michael Owen Krawitzky carried out a research internship for six months in a foreign institution: Institute National de la Recherche Agronomique (INRA) in Jouy-en-Josas (France) during 2012. As an additional requirement to qualify for the international mention, this thesis has been positively informed by two PhD experts currently working in a foreign research institution: Dr. Laura Soler (INRA, Val de la Loire, France) and Dr. Jessica Torrion (University of Montana, USA). Table of Contents Preface .......................................................................................................................... 7 Abstract ....................................................................................................................... 11 Abbreviations .............................................................................................................. 13 Objectives .................................................................................................................... 15 Structure ...................................................................................................................... 17 General introduction .................................................................................................. 21 1. Apple Fruit ......................................................................................................... 21 1.1 Production ........................................................................................................ 22 1.2 Pathology .......................................................................................................... 25 1.3 Calcium Influence ............................................................................................ 25 2. Bitter Pit ............................................................................................................. 28 2.1 Reducing Bitter Pit Occurrence ..................................................................... 30 2.2 Bitter Pit Prediction Models ........................................................................... 31 2.3 Bitter Pit Reproducibility Studies .................................................................. 33 New approaches to study bitter pit disorder ............................................................... 33 3. Phenolic Compounds in Apples ........................................................................ 35 4. Analysis of Phenolic Compounds and Antioxidant Activity .......................... 39 4.1. Phenolic compounds ....................................................................................... 39 4.2 Antioxidant Activity ........................................................................................ 40 4.2.1. Folin-Ciocalteu .......................................................................................... 40 4.2.2. Ferric Reducing Ability of Plasma (FRAP) .............................................. 41 4.2.3. Photochemiluminescence (PCL) ............................................................... 42 5. Study of proteins in biological systems ............................................................ 43 5.1 One-dimensional (1D) Gel Electrophoresis ................................................... 43 5.2 Two-dimensional (2D) Gel Electrophoresis .................................................. 45 6. Specialized Techniques ..................................................................................... 48 6.1 Apple Genome .................................................................................................. 48 6.2 Breeding ............................................................................................................ 49 6.3 Proteomics ........................................................................................................ 50 6.3.1 Bottom-up Proteomics ................................................................................ 52 6.3.2 Shotgun Proteomics .................................................................................... 53 Chapter 1 ..................................................................................................................... 63 Abstract .................................................................................................................. 64 Introduction ........................................................................................................... 65 Results and discussion ........................................................................................... 72 Conclusions ............................................................................................................ 79 Chapter 2 ..................................................................................................................... 91 Abstract .................................................................................................................. 92 Materials and Methods ......................................................................................... 96 9 Quantification of phenolic compounds by HPLC with ultraviolet–visible and ion trap mass spectrometry detection (HPLC-UV-Vis/MS) .............................. 98 Results and Discussion ........................................................................................ 101 Conclusions .......................................................................................................... 106 Chapter 3 ................................................................................................................... 115 Abstract ................................................................................................................ 116 Introduction ........................................................................................................