Scuola Dottorale di Ateneo Graduate School Dottorato di ricerca in Scienze Ambientali Ciclo XXVII Anno di discussione 2015 Chemical characterization of plant materials and development of analytical methodologies for metabolite determination SETTORE SCIENTIFICO DISCIPLINARE DI AFFERENZA: CHIM/01 Tesi di Dottorato di Elisa Scalabrin, matricola 825868 Coordinatore del Dottorato Tutore del Dottorando Prof. Gabriele Capodaglio Prof. Gabriele Capodaglio A Luca e ai nostri sogni Contents SUMMARY ............................................................................................................ 6 1 GENERAL INTRODUCTION ............................................................................. 8 1.1 Primary and secondary metabolism: Biosynthesis, metabolite typologies and functions ............... 10 1.1.1 Plant primary metabolism overview ............................................................................................. 10 1.1.1 The shikimate pathway ................................................................................................................. 12 1.1.2 Secondary metabolites: main classes of interest and functions ................................................... 12 1.1.2.1 Phenolics .................................................................................................................................... 13 1.1.2.2 Terpenoids ................................................................................................................................. 14 1.1.2.3 Alkaloids ..................................................................................................................................... 15 1.2 Chemical characterization of plants: objectives, analytical methodologies .................................... 16 1.3 Metabolomic approach: goals and potential ................................................................................. 17 1.3.1 Main analytical technique: HPLC-LTQ-Orbitrap ............................................................................ 19 1.3.2 Tools for metabolomic data analysis and metabolite identification ............................................. 19 1.3.2.1 Data processing ......................................................................................................................... 19 1.3.3 Molecule identification ................................................................................................................. 20 1.3.4 Statistical analysis .......................................................................................................................... 21 1.4 Thesis aims and research overview ............................................................................................... 22 2 INTEGRATION OF TARGETED AND UNTARGETED METABOLITE ANALYSES FOR STRESS RESPONSE COMPREHENSION: NICOTIANA LANGSDORFII AS A PLANT MODEL .................................................................................................... 24 2.1 Introduction .................................................................................................................................. 24 2.2 Materials and methods ................................................................................................................. 26 2.2.1 Sample preparation ....................................................................................................................... 26 2.2.1.1 Plant growth .............................................................................................................................. 26 2.2.1.2 Genetic modifications ................................................................................................................ 26 2.2.1.3 Stress inductions ........................................................................................................................ 26 2.2.2 Analysis of shikimic acids, jasmonic acid and salicylic acid ........................................................... 27 2.2.2.1 Theoretical definitions of quality control parameters ............................................................... 28 2.2.2.2 Internal standard and quantification ......................................................................................... 29 2.2.2.3 Sample treatment procedure .................................................................................................... 30 2.2.2.4 Instrumental method ................................................................................................................. 30 2.2.3 Analysis of total fatty acids ............................................................................................................ 31 2.2.3.1 Sample treatment procedure .................................................................................................... 31 2.2.3.2 Instrumental method and quantification .................................................................................. 32 2.2.4 Metabolomic analysis .................................................................................................................... 32 2.2.4.1 Sample treatment procedure .................................................................................................... 32 2.2.4.2 Instrumental method ................................................................................................................. 32 2.2.4.3 Data processing ......................................................................................................................... 34 2.3 Results .......................................................................................................................................... 34 2.3.1 Development of a quantitative methodology for the analysis of Shikimic, Salicylic and Jasmonic acids in Nicotiana Langsdorfii ...................................................................................................................... 34 2.3.1.1 Instrumental methodology ........................................................................................................ 34 2.3.1.2 Method validation and evaluation of matrix effect................................................................... 37 2.3.2 Analysis of Shikimic, Jasmonic and Salicylic acids in Nicotiana Langsdorfii .................................. 40 2.3.3 Analysis of fatty acids in Nicotiana Langsdorfii ............................................................................. 44 2.3.4 Metabolomic profile of Nicotiana Langsdorfii plants ................................................................... 46 2.3.5 Heat stress ..................................................................................................................................... 47 2.3.6 Chemical and water stress ............................................................................................................ 51 2.4 Discussion ..................................................................................................................................... 54 2.4.1 SA, SHA, JA validated method ....................................................................................................... 54 2.4.2 Fatty acid semi-quantitative method ............................................................................................ 55 2.4.3 Metabolite analysis ....................................................................................................................... 55 2.5 Conclusions ................................................................................................................................... 58 3 METABOLOMIC ANALYSIS OF GLYCYRRHIZA: CHARACTERIZATION OF SPECIES PECULIARITIES AND VARIETY SPECIFICITIES .................................. 60 3.1 Introduction .................................................................................................................................. 60 3.2 Materials and methods ................................................................................................................. 61 3.2.1 Sample preparation ....................................................................................................................... 61 3.2.2 Sample treatment procedure ........................................................................................................ 61 3.2.3 Instrumental method .................................................................................................................... 61 3.2.4 Data processing ............................................................................................................................. 61 3.3 Results .......................................................................................................................................... 62 3.3.1 Selection of the extraction solvent ............................................................................................... 62 3.3.2 Main constituents of Glycyrrhiza roots ......................................................................................... 63 3.3.3 Characteristic metabolites for species and variety ....................................................................... 67 3.4 Discussion ..................................................................................................................................... 70 3.5 Conclusions ................................................................................................................................... 72 4 METABOLOMIC ANALYSIS OF COFFEA ARABICA