The Role of the Chloroplast Outer Envelope in Jasmonate Synthesis der - - vorgelegt von Li Guan 2019 Erstgutachter: Prof. Dr. J. Soll Zweitgutachter: Prof. Dr. Peter Geigenberger Tag der Abgabe: 29.01.2019 Tag der mündlichen Prüfung: 19.03.2019 2 Zusammenfassung Table of Contents Summary ............................................................................................................................ 5 Zusammenfassung .............................................................................................................. 6 Abbreviations ...................................................................................................................... 7 1. Introduction .................................................................................................................... 9 1.1 Introduction of Jasmonates .................................................................................................. 9 1.2 Biosynthesis of JA ............................................................................................................... 10 1.3 Metabolism of JA ................................................................................................................ 12 1.4 The Bet v1-like superfamily ................................................................................................ 16 1.5 START domain containing proteins ................................................................................... 17 1.6 Aim of this work ................................................................................................................. 18 2.Material and Methods .................................................................................................... 20 2.1 Material .............................................................................................................................. 20 2.1.1 Chemicals ............................................................................................................................................ 20 2.1.2 Enzymes and Kits ................................................................................................................................ 20 2.1.3 Molecular weight and size markers ..................................................................................................... 21 2.1.4 Oligonucleotides .................................................................................................................................. 21 2.1.5 Vectors ................................................................................................................................................ 22 2.1.6 Bacterial strains ................................................................................................................................... 22 2.1.7 Antisera ............................................................................................................................................... 23 2.1.8 Bacteria and plants growth media and plates ...................................................................................... 23 2.1.9 Column material .................................................................................................................................. 23 2.1.10 Software ............................................................................................................................................ 23 2.1.11 Plant material and growth conditions ................................................................................................ 24 2.1.12 Wounding, cold and Pathogen treatment ........................................................................................... 24 2.2 Methods .............................................................................................................................. 25 2.2.1 Cloning ................................................................................................................................................ 25 2.2.2 Sequencing .......................................................................................................................................... 26 2.2.3 Genomic DNA isolation from Arabidopsis for genotyping PCR ........................................................ 27 2.2.4 RNA isolation from Arabidopsis leaves for cloning and real-time quantitative PCR ......................... 27 2.2.5 cDNA Synthesis .................................................................................................................................. 27 2.2.6 qRT-PCR (Quantitative Real-Time PCR) ........................................................................................... 27 2.3 Biochemical methods .......................................................................................................... 28 2.3.1 Protein overexpression in RosettaII cells ............................................................................................ 28 2.3.2 Purification of soluble overexpressed Jassy protein from soluble proteins ......................................... 28 2.3.3 Purification of Jassy out of inclusion bodies ....................................................................................... 29 2.3.4 Measuring the concentration of proteins ............................................................................................. 29 2.3.5 Isolation and fractionation of Arabidopsis thaliana chloroplast .......................................................... 30 2.3.7 Isolation of outer and inner envelope membranes from Pisum sativum .............................................. 30 2.3.7 In Vitro transcription ........................................................................................................................... 31 2.3.8 In Vitro translation ............................................................................................................................... 31 2.3.9 Chloroplast in vitro Import from Pisum sativum ................................................................................. 31 3 Zusammenfassung 2.3.10 Stable transformation of Arabidopsis ................................................................................................ 32 2.3.11 SDS polyacrylamide gel electrophoresis (SDS-PAGE) .................................................................... 32 2.3.12 Western blotting ................................................................................................................................ 33 2.3.13 Immunodetection ............................................................................................................................... 33 2.3.15 Transient transformation of Nicotiana benthamiana ......................................................................... 34 2.3.16 Protoplast isolation from Nicotiana benthamiana ............................................................................. 34 2.3.17 Microscale Thermophoresis .............................................................................................................. 34 2.3.18 Carboxyfluorescein Assay ................................................................................................................. 35 2.3.19 Electrophysiology Assay ................................................................................................................... 35 2.3.20 Jasmonate Measurements .................................................................................................................. 37 3. Results .......................................................................................................................... 39 3.1 JASSY is a novel protein with a conserved START domain .............................................. 39 3.2 JASSY is localized to the chloroplast outer membrane ...................................................... 40 3.3 Phenotypic analysis of the jassy mutant .............................................................................. 46 3.3.1 Root growth in the jassy mutant is slightly decreased ......................................................................... 46 3.3.2 Loss of JASSY increases susceptibility to pathogen attack ................................................................ 49 3.3.3 Loss of JASSY decreases cold tolerance in Arabidopsis. ................................................................... 51 3.4 JASSY is involved in the JA biosynthesis pathway ............................................................ 52 3.4.1 The expression of JA-responsive gene (PDF1.2) was not activated in the jassy mutant upon pathogen attack ............................................................................................................................................................ 52 3.4.2 The expression of JA-responsive gene (ICE1) was not activated in the jassy mutant upon cold treatment ....................................................................................................................................................... 53 3.4.3 The expression of JA responsive genes is not activated in the jassy mutant upon wounding ............. 54 3.4.4 Feeding the jassy mutant with JA-Ile or JA biosynthesis intermediate, OPDA, restored the induction of JA-responsive gene PDF1.2..................................................................................................................... 57 3.4.5 Lack of JASSY prevented JA accumulation ......................................................................................