The substrate specificity of N-end rule components from Arabidopsis thaliana Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Frau Maria Katharina Klecker geb. am 03.10.1986 in Marburg Gutachter Prof. Dr. Ingo Heilmann, Martin-Luther-Universität Halle-Wittenberg Prof. Dr. Nico Dissmeyer, Universität Osnabrück Prof. Dr. Frederica Theodoulou, Rothamsted Research, Harpenden Verteidigung 18. Dezember 2019 Table of contents Table of contents Table of contents ...................................................................................................................................... I Abbreviations .......................................................................................................................................... V Summary ................................................................................................................................................ IX Zusammenfassung ................................................................................................................................... X 1. Introduction ..................................................................................................................................... 1 1.1 Protein degradation in plants .................................................................................................. 1 1.1.1 Bulk protein degradation ................................................................................................. 1 1.1.2 Selective protein degradation – the 26S proteasome ..................................................... 1 1.1.3 Ubiquitin .......................................................................................................................... 2 1.1.4 Plant E3 ligases ................................................................................................................ 4 1.2 The N-end rule pathway of protein degradation .................................................................... 7 1.2.1 Functions of the N-end rule pathway .............................................................................. 9 1.2.2 Primary destabilizing amino acids and N-recognins ...................................................... 10 1.2.3 Secondary destabilizing N-termini ................................................................................ 15 1.2.4 Tertiary destabilizing N-termini ..................................................................................... 16 1.3 Male gametogenesis in Arabidopsis ...................................................................................... 18 1.3.1 Regulation of pollen development ................................................................................ 21 1.4 The phytohormone ethylene................................................................................................. 26 1.4.1 Impact of ethylene on plant physiology ........................................................................ 26 1.4.2 Ethylene signaling .......................................................................................................... 28 1.5 The phytohormone cytokinin ................................................................................................ 32 1.5.1 Cytokinin during etiolation ............................................................................................ 33 2. Aims and scope of the thesis ......................................................................................................... 35 3. Results ........................................................................................................................................... 37 3.1 The genetic background in prt1-1ts confers thermosensitivity of pollen development ........ 37 3.1.1 The prt1-1ts line is sterile at an elevated temperature ................................................. 37 3.1.2 The thermosensitivity of prt1-1ts is independent of the PRT1 locus ............................. 38 3.1.3 The heat-induced sterility of prt1-1ts is caused by a defect in male gametogenesis .... 40 3.1.4 Microspore to pollen transition at 28°C is affected in prt1-1ts...................................... 43 3.1.5 Conditional male sterility of prt1-1ts is likely a sporophytic trait .................................. 46 3.1.6 Several genes are differentially expressed in anthers of prt1-1ts .................................. 47 3.2 PRT1 promoter activity .......................................................................................................... 50 3.3 Biochemical characterization of the PRT1 protein ................................................................ 53 I Table of contents 3.3.1 PRT1 acts as an E3 ubiquitin ligase in vitro.................................................................... 54 3.3.2 Both RING domains of PRT1 are required for autoubiquitination ................................ 56 3.3.3 The in-vitro substrate specificity of PRT1 ...................................................................... 59 3.3.4 PRT1 RING domains are not involved in substrate binding in vitro .............................. 62 3.3.5 In-vitro PRT1 binding to Arabidopsis peptide sequences .............................................. 64 3.4 EIN2 is a potential in-vivo substrate for PRT1 ....................................................................... 66 3.4.1 EIN2C-term is destabilized in a PRT1-dependent manner in vivo ..................................... 67 3.4.2 Apparent molecular weight of EIN2C-term ....................................................................... 73 3.4.3 Physical interaction between EIN2 and PRT1 in vivo .................................................... 73 3.4.4 Hypocotyl elongation in the dark is slightly increased in prt1-1ts ................................. 76 3.4.5 Cytokinin-induced inhibition of hypocotyl elongation in the dark is impaired in prt1-1ts and N119 ....................................................................................................................................... 78 3.4.6 Expression of EIN2C-term confers signs of constitutive ethylene response in combination with the prt1-1ts genotype ............................................................................................................ 84 3.5 The substrate specificities of PRT6 and ATE1 ........................................................................ 86 3.5.1 The in-vitro substrate specificity of the UBR domain of PRT6 ...................................... 86 3.5.2 ATE1 arginylates acidic N-termini in vitro ..................................................................... 90 4. Discussion ...................................................................................................................................... 93 4.1 A new temperature-dependent sterility phenotype ............................................................. 93 4.1.1 Potential origin of the sterility phenotype in prt1-1ts ................................................... 93 4.1.2 The thermosensitivity of prt1-1ts reproduction ............................................................. 96 4.2 PRT1 acts as an E3 ligase in-vitro using both RING domains ............................................... 103 4.3 The in-vitro substrate specificity of plant N-recognins ....................................................... 106 4.3.1 Determinants of N-degron recognition by PRT1 ......................................................... 106 4.3.2 BB is an in-vivo substrate of PRT1 ............................................................................... 110 4.3.3 In-vitro substrate interactions of PRT6 ........................................................................ 112 4.3.4 In-vitro arginylation activity of ATE1 ........................................................................... 114 4.4 A potential role for PRT1 in EIN2 signaling .......................................................................... 116 4.4.1 Physical interaction of EIN2 and PRT1 ......................................................................... 116 4.4.2 EIN2C-term has low in-vivo protein stability ................................................................... 117 4.4.3 In-vivo stability of EIN2C-term depends on PRT1 activity ............................................... 118 4.4.4 EIN2C-term degradation in planta .................................................................................. 121 4.5 The genetic background of prt1-1 lines confers cytokinin hyposensitivity in the dark ...... 123 4.5.1 Sugar-dependence of cytokinin hyposensitivity in prt1-1ts/N119 ............................... 124 5. Conclusions .................................................................................................................................. 128 II Table of contents 6. Materials and Methods ............................................................................................................... 130 6.1 Plant growth conditions ...................................................................................................... 130 6.2 Plant material ...................................................................................................................... 130 6.3 Hormone treatments ........................................................................................................... 131