Characterization of STRUBBELIG, an atypical receptor-like kinase, and components of its signaling pathway in Arabidopsis [Elektronische Ressource] / Martine Batoux

Martine Batoux

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Biologikum Weihenstephan
Fachgebiet für Entwicklungsbiologie der Pflanzen
Technische Universität München

Characterization of STRUBBELIG, an Atypical Receptor-
Like Kinase, and
Components of its Signaling Pathway in Arabidopsis

Martine Batoux

Vollständiger Abdruck der von der Fakultät
Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
der Technischen Universität München
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.

Vorsitzender: Univ-Prof. Dr. Erwin Grill
Prüfer der Dissertation: 1. Univ-Prof. Dr. Kay H. Schneitz
2. Univ-Prof. Dr Alfons Gierl

Die Dissertation wurde am 12. Februar 2007 bei der Technischen Universität
München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt am 20. März 2007 angenommen.TABLE OF CONTENTS
Table of Contents
SUMMARY ________________________________________________________________ 5
ZUSAMMENFASSUNG_______________________________________________________ 6
1-1 CELL FATE ____________________________________________________ 8
1-1-1 Importance of cell position ____________________________________________ 8
1-1-2 Cell identity________________________________________________________ 9
1-2 ORGAN SHAPE_______________ 12
1-2-1 Cell division plan __________________________________________________ 12
1-2-2 Cell elongation ____________________________________________________ 13
1-2-2-1 Microfibrils deposition in the cell wall _______________________________ 13
1-2-2-2 Cortical microtubule array ________________________________________ 14
1-3 STRUBBELIG, A RECEPTOR LIKE-KINASE INVOLVED IN PLANT DEVLOPMENT _ 14
1-3-1 Phenotype of sub __________________________________________________ 14
1-3-2 SUB, a receptor like-kinase (RLK) _____________________________________ 15
1-3-3 Role attributed to SUB ______________________________________________ 16
1-3-4 SUB may act non-cell autonomously ___________________________________ 16
1-3-5 The aim of this work ________________________________________________ 16
1-4 REFERENCES_________________________________________________ 17
Chapter 2: Structure/Function analysis of STRUBBELIG _________________________ 21
2-1 INTRODUCTION_______________ 21
2-1-1 The extracellular part of LRR-RLKs ____________________________________ 22
2-1-1-1 LRR domains _________________________________________________ 22
2-1-1-2 Cysteine pairs _________________________________________________ 22
2-1-1-3 Other extracellular domains ______________________________________ 23
2-1-2 The transmembrane domain _________________________________________ 24
2-1-3 The intracellular domain_____________________________________________ 25
2-2 MATERIALS AND METHODS ____________________________________ 27
2-2-1 Plant work________________________________________________________ 27
2-2-2 Scanning electron microscopy ________________________________________ 28
2-2-3 Forward genetic screen _____________________________________________ 28
2-2-4 Reverse genetic approaches _________________________________________ 28
2-2-4-1 T-DNA insertion lines ___________________________________________ 28
2-2-4-2 TILLING______________________________________________________ 29
2-2-5 Site-directed mutagenesis ___________________________________________ 30
2-2-5-1 Generation of SUB overexpression construct_________________________ 30
2-2-5-2 Generation of mutagenized variants ________________________________ 31
2-3 RESULTS_____________________________________________________ 32
2-3-1 Isolation of new alleles of SUB________________________________________ 32
2-3-1-1 EMS mutagenesis______________________________________________ 32
2-3-1-2 TILLING______________________________________________________ 34
2-3-1-3 Characterization of T-DNA lines ___________________________________ 35
2-3-2 Phenotypic analysis of sub alleles _____________________________________ 37
2-3-2-1 Comparison of sub phenotype in Ler background _____________________ 37
2-3-2-2 T-DNA sub lines phenotype ______________________________________ 22
2-3-3 Site-directed mutagenesis approach ___________________________________ 23
2-3-3-1 Rescue of sub-1 phenotype by a 35S::SUB transgene _________________ 24
2-3-3-2 Role of C57 and C365-6 in SUB function ____________________________ 24
2 TABLE OF CONTENTS
2-3-3-3 Does SUB kinase domain active?__________________________________ 25
2-3-3-4 is phosphorylation of SUB important for its function? ___________________ 26
2-4 DISCUSSION__________________________________________________ 28
2-4-1 Role of different SUB AAs ___________________________________________ 28
2-4-4-1 AA with an inconsiderable role in SUB function _______________________ 28
2-4-4-2 AAs with a considerable role in SUB function_________________________ 29
2-4-2 Phenotypic comparison of different sub alleles ___________________________ 33
2-4-2-1 Different alleles of sub in Ler show the same strength of phenotype _______ 33
2-4-2-2 Natural variations may affect sub phenotype _________________________ 34
2-5 REFERENCES_________________ 35
Chapter 3: STRUBBELIG, a gene expressed in proliferate tissues_________________ 40
3-1 INTRODUCTION _______________________________________________ 40
3-2 MATERIALS AND METHODS____ 42
3-2-1 SUB::GUS reporter line generation ____________________________________ 42
3-2-2 Detection of GUS activities___________________________________________ 43
3-2-3 Tissue preparations for microscopy ____________________________________ 43
3-2-3-1 Shoot apical meristems and flowers ________________________________ 43
3-2-3-2 Seedling roots _________________________________________________ 44
3-2-2-4 Microscopy and artwork _________________________________________ 44
3-3 RESULTS_____________________________________________________ 44
3-3-1 Correlation between SUB::GUS line and SUB ISH results __________________ 45
3-3-2 Analysis of the GUS activity pattern, reflecting SUB expression ______________ 47
3-3-2-1 Expression of SUB::GUS during vegetative phase_____________________ 47
3-3-2-2 Expression of SUB::GUS during reproductive phase ___________________ 51
3-4 DISCUSSION__________________ 53
3-4-1 SUB may involve in lateral roots and leaf vascular system development _______ 54
3-4-2 SUB is expressed in dividing tissues ___________________________________ 55
3-4-3 Possible role of SUB in dividing tissue__________________________________ 56
3-5 REFERENCES_________________________________________________ 56
Chapter 4: Identification of putative interacting partners of SUB __________________ 59
4-1 INTRODUCTION_______________ 59
4-2 MATERIALS AND METHODS ____________________________________ 64
4-2-1 cDNA isolation of ALADIN ___________________________________________ 64
4-2-2 Non-radioactive in situ hybridization ___________________________________ 65
4-2-3 Computational analysis of ALADIN ____________________________________ 66
4-2-4 Yeast two-hybrid __________________________________________________ 67
4-2-4-1 Yeast strains __________________________________________________ 67
4-2-4-2 Plasmid constructs _____________________________________________ 67
4-2-4-3 Yeast transformation and verification of the interaction between SUB and some
candidates in yeast ___________________________________________________ 68
4-2-4-4 Rescue of yeast plasmid DNA from positives clones ___________________ 69
4-2-5 Expression of protein and in vitro pull down _____________________________ 69
4-2-5-1 Plasmid constructs _____________________________________________ 69
4-2-5-2 Expression and purification of GST-SUB in E.Coli ___________________ 69 intra
4-2-5-3 Transcription/translation of radiolabelled proteins _____________________ 71
4-2-5-4 In vitro pull down assays_________________________________________ 72
3 TABLE OF CONTENTS
4-3 RESULTS_____________________________________________________ 72
4-3-1 identification of putative partners of STRUBBELIG ________________________ 72
4-3-1-1 Two-hybrid interaction of SUB and the putative partner _________________ 72
4-3-1-2 Structure and characterization of the candidates ______________________ 75
4-3-2 STRUBBELIG interacts in vitro with a putative nucleoporin, ALADIN __________ 77
4-3-2-1 Expression pattern of ALADIN ____________________________________ 78
4-3-2-2 ALADIN belong to WD40 family ___________________________________ 79
4-3-2-3 in vitro interaction between SUB and ALADIN ________________________ 84
4-4 DISCUSSION__________________________________________________ 85
4-4-1 The different putative candidates ______________________________________ 85
4-4-1-1 Candidates with a known role in RLKs pathway_______________________ 85
4-4-1-2 Candidates with a role in signaling _________________________________ 87
4-4-1-3 Candidates related to cytoskeleton_________________________________ 89
4-4-2 Interaction of SUB and ALADIN _______________________________________ 91
4-4-2-1 Structure of ALADIN ____________________________________________ 91
4-4-2-2 SUB interacts with ALADIN in yea