Molecular and cell biological analysis of the ESCRT system in Arabidopsis thaliana [Elektronische Ressource] / vorgelegt von Mojgan Shahriari

Molecular and cell biological analysis of the ESCRT
system in Arabidopsis thaliana





Inaugural-Dissertation



zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln





vorgelegt von


Mojgan Shahriari



Aus Iran




Köln 2008







I





















Berichterstatter: Prof. Dr. Martin Hülskamp
Prof. Dr. Ute Höcker



Prüfungsvorsitzender: Prof. Dr. Siegfried Roth





Tag der mündlichen Prüfung: November 2008










IIAcknowledgment
It is a heart warming and rewarding experience to pay tribute to the people whose
invaluable contributions helped me through out my time as a PhD researcher at cologne
university, Köln.
My sincere and profound gratitude goes to Prof. Martin Hülskamp how gave me a chance
to be a member of Botanical Institute III and Dr. Swen SChellmann for giving me an
opportunity to join his group and for the trust that he put into me. It was a unique
experience to work with him. His excellent scientific guidance helped me expanded my
capabilities.
My special thanks go to my thesis Committee, Prof. Dr. Martin Hülskamp, Prof. Dr. Ute
Höcker and Prof. Dr. Siegfried Roth.
I would like to acknowledge the assistance of the technicians of the department: Birgit
Kernebeck, Britta Müller, Irene Klinkhammer, Bastian Welter and Uschi Claßen. The
personal encouragement and actual help I received from them has made it possible to
complete my studies during my time in the University of Cologne.
My special thanks go to the members of the ELCH Group: Christoph Spitzer, Aneta
Sabovljevice, Channa Keshavaiah, Stefanie Herbeth, Florian Heßner, and Britta Müller
for their extensive for nice comments and the many discussions and for creating a
pleasant working atmosphere.
I am indebted to Dr. Schellmann, Dr. Roodbarkelari, Dr. Pesch, Dr. Horvath and Dr.
Digiuni for the critical reading of the whole text and for the valuable contribution to the
clearness of the manuscript.
And many thanks go to Katja Wester for her help on Confocal Microscopy
I would like to thank Ullrich Herrmann for his help on computer tasks e to thank Niko Pawlowski and Rainer Häusler for helping me run the protein
experiments.
I want to thank Ilona Zimmermann, Joachim Uhrig for helping me run yeast two hybrid
technique.
My special thanks go to Gardeners of Botanic Institute, who prepared excellent plants for
my research.
IIII would like to thank Simona, Channa, Aneta, Anderea, Jenny, Alex, Nora for creating
such a great friendship at the office.
There are all the other people who have made Botanic III very special place over all those
years: Andrea Schrader, Burcu Dartan, Cho-Chun Huang, Elena Galiana Jaime, Ullrich
Herrmann, Marc Jakoby, Cordula Jörgens, Rachappa Balkunde, Valerie Mach, Philipp
Thomas, Elena Galiana Jaime, Moola Mutondo, Victor Kirik, Angela Kirik, Rainer
Saedler, Daniel Bouyer, and all the others who have passed through.
I would like to express my thanks to all members of Botanic III Institute who have helped
me directly and indirectly in accomplishing this project and giving me a leaning
environment to grow me personally as well as professionally.
I appreciated from SFB 635 for their financial support of my study.
I would like to thank everybody who was important to the successful realization of thesis,
as well as expressing my apology that I could not mention personally one by one.
I cannot finish without saying how grateful I am with my family. My husband, Farshad,
my daughter, Dorsa, my sister and my brothers all have given me a loving environment
where to develop.
Finally, and most importantly, I wish to thank my parents, MohamadAli and Sakineh.
They have always supported and encouraged me to do my best in life. To them I dedicate
this thesis.









IV
CONTENTS
Zusammenfassung.................................................................................................................III
Abstract…………………...…………………………………………………………….......V
Publications.........................................................................................................................VII
Abbreviations and Genename............................................................................................VIII
Figure index …………….………………………………………………………………....IX

1. Introduction
1.1 Endomembrane system in eukaryotes………………………………………………..1
1.2 Trafficking in the ESCRT system………………………………………………........3
1.2.1 The ESCRT pathway in plants……………………………………………………..5
1.2.2 Vps4 is an important subunit in ESCRT machinery……………...………………..6
1.3 Model systems to study the function of the ESCRT in plants………………….........8
Aim………………………………………………………………………………….....10

2. Results
2.1 The ESCRT complex network in Arabidopsis thaliana…………………………….11
2.2 Interaction assay of Arabidopsis putative ESCRT proteins………………………....11
2.2.1 Yeast two hybrid assay as a technique to show the interactions of the
Arabidopsis putative ESCRT proteins…………………………………………………..11
2.2.2 Split–YFP was used to prove the Yeast two hybrid data………………………….12
2.2.3 ESCRT I Complex in Arabidopsis thaliana ……………………………………....14
2.2.4 Interaction assay of ESCRT II using Yeast two hybrids and Split YFP…………..15
2.2.5 Interaction assay of ESCRT III using Yeast two hybrids…………………………16
2.2.6 Protein interaction of putative upstream components and ESCRT-I by Yeast
two-hybrid and Bimolecular Florescent Complementation assay………………………17

2.2.7 Interaction between ESCRT I, ESCRT II and ESCRT III components………......19
2.2.8 Downstream ESCRT proteins interact with each other and the ESCRT III……....19
2.2.9 Arabidopsis ESCRT components are interacting on endosomes………………….20
2.3 Characterization and functional analysis of AtSKD1…………………………….....24
+2.3.1 Identification of AtSKD1/Vps4 (Suppressor of K TRANSPORT GROWTH
DEFFECT1) homologue in Arabidopsis…………………………………………….......24
2.3.2 AtSKD1/Vps4 interacts with its upstream and downstream proteins……………..24
2.3.3 ATPase assay of AtSKD1………………………………………………………....30
2.3.4 AtSKD1 colocalizes with the endosomal Ara6 and Ara7 markers on MVB….......33
2.3.5 Expression of AtSKD1 in Arabidopsis plants……………………………………..35
2.3.5.1 Localization of AtSKD1 in root………………………………………………....35
2.3.5.2 Ectopic expression of AtSKD1 and its dominant negatives in trichomes……....37
2.3.5.3 Localization of AtSKD1 and its dominant negatives variants in trichomes…….39
2.3.6 Mutation in AtSKD1 reveals vacuole defection in trichome………………….......42

3. Discussion
3.1 The ESCRT pathway in Arabidopsis thaliana………………………………..……..45
V3.2 The MVB pathway is highly conserved during the eukaryotic development ….......45
3.3 The role of AtSKD1 in the MVB pathway in Arabidopsis thaliana………………...47
3.3.1 AtSKD1 is a functional homolog of Vps4 in Arabidopsis thaliana……………….48
3.3.2 AtSKD1 functions in the protein sorting pathway in Arabidopsis………………...49
3.4 Cytokinesis needs a functional AtSKD1…………………………………………….50
3.5 Ectopic expression of AtSKD1 induces the vacuolar fragmentation and cell death...51
3.5.1 Vacuole maintenance is defected in the AtSKD1 mutants………...……………....51
3.5-2 Cell death; the last chapter of AtSKD1 mutation………………………….………53


4. Material and Methods
4.1 Material
4.1.1 Chemicals, antibiotics……………………………………………………………..55
4.1.2 Enzymes and molecular biological materials……………………………………..55
4.1.3 Cloning vectors……………………………………………………………………55
4.1.4 Antibiotics…………………………………………………………………………56
4.1.5 Bacterial strains……………………………………………………………………56
4.1.6 Plant lines………………………………………………………………………….56

4.2 Methods
4.2.1 Plant work ………………………………………………………………………...57
4.2.1.1 Plant growth conditions……………………………………………………........57
4.2.1.2 Plant transformation………………………………………………………….....57
4.2.1.3 Selection of transformants……………………………………………………....57
4.2.1.4 Seed sterilization……………………………………………………………......58
4.2.1.5 DAPI staining…………………………………………………………………...58
4.2.1.6 Propidium iodide staining…………………………………………………….....58
4.2.1.7 Fluoresceine diacetate staining………………………………………………….58
4.2.1.8 Microscopy……………………………………………………………………...58
4.2.2 Molecular-biological methods…………………………………………………….59
4.2.2.1 Genomic DNA preparation from plant tissue…………………………………...59
4.2.2.2 Plasmid DNA preparation from bacteria………………………………………..59
4.2.2.3 DNA-manipulation……………………………………………………………...59
4.2.2.4 Plasmid construction………………………………………………………….....60
4.2.2.5 BiFC constructs……………………………………………………………….....60
4.2.2.6 Protoplast prepearation and transformation…………………………………......61
4.2.2.7 Yeast two-hybrid assay……