Influence of post-anthesis drought stress on germination and malting quality in barley Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Korana Surdonja geb. am: 17.04.1986 in: Koper, Slowenien Gutachter: 1. Prof. Dr. Thomas Altmann 2. Prof. Dr. Klaus Humbeck 3. Prof. Dr. Wolfgang Dröge-Laser Datum der Verteidigung: 06.03.2019 Contents List of figures ............................................................................................................................... i List of tables ............................................................................................................................... iii Abbreviation ........................................................................................................................... iv 1 Introduction .............................................................................................................................. 1 1.1 Characteristics of malting barley grain .............................................................................. 1 Endosperm ........................................................................................................................... 2 Aleurone layer ..................................................................................................................... 5 Embryo ................................................................................................................................ 6 Husk ..................................................................................................................................... 6 1.2 Malting process ................................................................................................................. 7 Steeping ............................................................................................................................... 7 Germination ......................................................................................................................... 9 Kilning ................................................................................................................................. 9 1.3 Malting quality parameters .............................................................................................. 10 1.4 Molecular mechanism behind the dormancy release ....................................................... 12 1.5 Translational initiation and role of stored messenger RNA upon imbibition ................. 14 1.6 Effects of terminal drought stress on dormancy and germination ................................... 15 1.7 Aim of the study .............................................................................................................. 16 2 Materials and methods ............................................................................................................ 18 2.1 Plant material ................................................................................................................... 18 2.2 Plant cultivation and sampling ........................................................................................ 18 2.3 Phenotyping ..................................................................................................................... 19 2.3.1 Evaluation of yield and grain traits .......................................................................... 19 2.3.2 Germination assays ................................................................................................... 20 2.4 Micromalting ................................................................................................................... 20 2.5 Biochemical analysis ....................................................................................................... 21 2.5.1 Carbon and nitrogen analysis of mature seeds ......................................................... 21 2.5.2 Starch measurement .................................................................................................. 21 2.6 Phytohormone analysis .................................................................................................... 21 2.6.1 Abscisic acid, cytokinin, auxin and salicylic acid extraction ................................... 22 2.6.2 Gibberellic acid extraction ....................................................................................... 22 2.6.3 Quantification of phytohormones by Electrospray Ionization Tandem Mass Spectrometry ...................................................................................................................... 23 2.7 Transcriptome analysis .................................................................................................... 23 2.7.1 Polysomal RNA extraction ....................................................................................... 23 2.7.2 RNA extraction from sucrose fractions .................................................................... 25 2.7.3 Total RNA extraction ............................................................................................... 25 2.7.4 Quality checking of the RNA using Agilent 2000 Bioanalyzer ............................... 26 2.7.5 Synthesis of complementary DNA ........................................................................... 26 2.7.6 Quantitative real-time polymerase chain reaction (qRT-PCR) ................................ 27 2.7.7 Microarray hybridization .......................................................................................... 27 2.7.8 Microarray data analysis ........................................................................................... 27 2.7.9 Gene ontology enrichment analysis .......................................................................... 28 2.8 QTL analysis and statistics .............................................................................................. 28 3 Results .................................................................................................................................... 30 3.1 Two contrasting barley elite lines differ in their abscisic acid content and germination behavior ................................................................................................................................. 30 3.2 Germination and shoot emergence of the DH population ............................................... 33 3.3 Relationship among traits ................................................................................................ 34 3.4 Germination and shoot emergence quantitative trait loci ................................................ 40 3.4.1 Co-localization of quantitative trait loci related to germination and shoot emergence with malting quality traits .................................................................................................. 42 3.5 Transcriptome analysis of LP104 and LP106 ................................................................. 44 3.5.1 Dry seed transcriptome ............................................................................................. 45 3.5.2 Transcriptome analysis of micromalted samples ..................................................... 51 3.5.3 Identification of transcripts important for early germination processes ................... 52 3.5.4 Comparison of total RNA and the polysome-bound RNA ....................................... 53 3.5.5 Gene ontology enrichment analysis .......................................................................... 54 3.6 Analysis of barley seed maturation and germination process using transgenically modulated ABA levels .......................................................................................................... 54 3.6.1 Confirmation of AtNCED6 transgene expression ..................................................... 55 3.6.2 Analysis of HvNCED and HvABA8´-OH family in Golden Promise and transgenic lines .................................................................................................................................... 55 3.7. Germination and shoot emergence of transgenic barley lines with modulated ABA content ................................................................................................................................... 57 3.8 Hormone content of developing grains with transgenic modulated ABA pathway ........ 61 3.8.1 Analysis of abscisic acid and its degradation products in developing transgenic seeds .................................................................................................................................. 61 3.8.2 Gibberellic acid ........................................................................................................ 65 3.8.3 Threshold of ABA:GA ratio as determinant for germination process ..................... 67 3.9 Transcriptome analysis during germination .................................................................... 69 3.9.1 Seed transcriptome of Golden Promise .................................................................... 70 3.9.2. Changes in the transcriptome of dry seed in dependence to ABA .......................... 72 3.9.3 Differences in the transcriptome during imbibition in Golden Promise .................. 76 3.9.4 Transcriptional changes during imbibition independence to ABA .......................... 78 3.9.5 Association of transcripts in the embryo and endosperm at 12 HAI with polysomes