Nonrandom mating in Nicotiana attenuata and the paternal influence on seed metabolomes and pathogen resistance Dissertation To Fulfill the Requirements for the Degree of „doctor rerum naturalium“ (Dr. rer. nat.) Submitted to the Council of the Faculty of Biology and Pharmacy of the Friedrich Schiller University Jena by M.Sc., Xiang Li born on 10th November, 1986 in Qingdao, Shandong, China 1. Gutachter: Prof. Dr. Ian T. Baldwin, Max Planck Institute for Chemical Ecology, Jena, Germany 2. Gutachter: Prof. Dr. Erika Kothe, Friedrich-Schiller-University, Jena, Germany 3. Gutachter: Associate Prof. Åsa Lankinen, Swedish University of Agricultural Sciences, Uppsala, Sweden Tag der öffentlichen Verteidigung: 26th. 09. 2018 Contents Contents Abbreviations ................................................................................................................................... III 1. Introduction ................................................................................................................................... 1 1.1 Sexual selection and its application in plants ......................................................................................... 1 1.2 Sexual selection in plants ........................................................................................................................ 2 1.3 Nonrandom mating among compatible donors in Nicotiana attenuata ................................................ 5 1.4 PT growth rates and stylar metabolites in nonrandom mating .............................................................. 8 1.5 Paternal effects on offspring qualities and seed metabolites .............................................................. 10 1.6 The interaction between seeds and soil microbes and roles of seed metabolites............................... 12 2. Aims of the thesis ......................................................................................................................... 17 3. Materials and methods ................................................................................................................ 18 3.1 In vivo pollen tube competition and secondary metabolites implicated in nonrandom mating of Nicotiana attenuata ............................................................................................................................... 18 3.1.1 Plant material and growth conditions .............................................................................................. 18 3.1.2 Emasculation and pollination ........................................................................................................... 18 3.1.3 Estimation of the number of pollen grains on one zone of the stigma in separated pollination .... 20 3.1.4 In vivo pollen tube visualization, length measurement and further data acquisition ..................... 20 3.1.5 Paternity assessment of matured seeds .......................................................................................... 22 3.1.6 Statistical analysis ............................................................................................................................ 22 3.1.7 Metabolomics analyses of pollinated styles .................................................................................... 23 3.1.8 Metabolite analyses on non-pollinated styles and in vitro PTs ....................................................... 25 3.2 Paternal effects on seed metabolomes and differential pathogen resistance ..................................... 26 3.2.1 Plant materials and hand pollination for hybrid seeds .................................................................... 26 3.2.2 Seed metabolite extraction and further metabolomics analyses .................................................... 28 3.2.3 Fungus isolation and culturing ......................................................................................................... 29 3.2.4 Fungal DNA extraction and identification ........................................................................................ 30 3.2.5 Fungal morphology observation ...................................................................................................... 31 3.2.6 Pathogen bioassays and dual culture assays ................................................................................... 32 3.2.7 Statistical analyses ........................................................................................................................... 34 I Contents 4. Results ......................................................................................................................................... 35 4.1 In vivo pollen tube competition and secondary metabolites implicated in nonrandom mating of N. attenuata ............................................................................................................................................... 35 4.1.1 Pollen performance showed no significant difference among single pollinations .......................... 35 4.1.2 The novel sample mounting method and the description of the two transmitting zones .............. 37 4.1.3 Two different mixed-pollination methods led to the same seed sets ............................................. 39 4.1.4 The differential in vivo PT growths of two pollen donors were involved in disequilibrium paternity .................................................................................................................................................................... 40 4.1.5 O-acyl sugars were found as potential mate selection-related metabolites ................................... 43 4.2 Paternal effects on seed metabolomes and differential pathogen resistance ..................................... 48 4.2.1 Metabolomic profiles of the hybrid seeds showed clear differences .............................................. 48 4.2.2 Selection of pathogens and different mortalities of different seeds ............................................... 50 4.2.3 Constitutive metabolites potentially contributing to differential pathogen resistance of seeds .... 53 4.2.4 Trichoderma harzianum inhibited the growth of the fungal pathogens ......................................... 56 5. Discussion .................................................................................................................................... 59 5.1 In vivo pollen tube growth rate and nonrandom mating in N. attenuata ............................................ 59 5.2 Secondary metabolites and nonrandom mating in N. attenuata ......................................................... 64 5.3 Paternal effects on seed metabolites and seed resistance to pathogens ............................................ 65 5.4 Biocontrol agents and seeds in seed banks .......................................................................................... 68 5.5 Outlook ................................................................................................................................................. 70 6. Summary ..................................................................................................................................... 73 7. Zusammenfassung........................................................................................................................ 76 8. Supplementary data ..................................................................................................................... 80 9. References ................................................................................................................................... 88 Eigenständigkeitserklärung ............................................................................................................ 108 Acknowledgement ......................................................................................................................... 109 II Abbreviations Abbreviations AMF Arbuscular mycorrhizal fungi CTAB Cetyltrimethylammonium bromide DNA Deoxyribonucleic acid ESI/HR-Q-TOF Electrospray ionization/ high resolution quadrupole time-of-flight ESI/TOF Electrospray ionization/ time-of-flight EV Empty vector GA3 Gibberellic acid GB5 Gamborg’s B5 medium GLM Generalized linear model GUS β -Glucuronidase ITS Internal transcribed spacer LSM Laser scanning microscope LSU Large ribosomal subunit MS Mass spectrometry PC Principal component PCA Principal component analysis PCD Programmed cell death PCR Polymerase chain reaction PDA Potato dextrose agar PPEB Post-pollination ethylene burst PrpS Papaver rhoeas pollen S PrsS Papaver rhoeas style S PT Pollen tube RNA Ribonucleic acid RNase Ribonuclease UHPLC Ultrahigh-performance liquid chromatography III Abbreviations IV Introduction 1. Introduction 1.1 Sexual selection and its application in plants In The Descent of Man (Darwin, 1871), Darwin adopted Hunter’s distinction between primary and secondary sexual characters (Hunter, 1780, 1837) and first coherently explained elaborate weapons and ornaments found in many male animals and less commonly in females. In his book, secondary sexual characters were defined by Darwin as characters used in intrasexual competition to breed. Darwin termed the evolutionary process generating those characters “sexual selection”. In his book (Darwin, 1871), he suggested two mechanisms of sexual selection: “In the one it is between individuals of the same sex, generally the males, in order to drive away or kill their rivals, the females remaining passive; whilst in the other, the struggle