Analysis of auxin and abscisic acid signal transduction in Arabidopsis thaliana [Elektronische Ressource] / Yulin Tang

Ronan Quilliou

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 16
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Institut für Botanik und Mikrobiologie
Lehrstuhl für Botanik
Technische Universität München




Analysis of auxin and abscisic acid signal
transduction in Arabidopsis thaliana






by
Yulin Tang



Institut für Botanik und Mikrobiologie
Lehrstuhl für Botanik
Technische Universität München

Analysis of auxin and abscisic acid signal transduction
in Arabidopsis thaliana

Yulin Tang

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. U. Schmidhalter
Prüfer der Dissertation: 1. Univ.- Prof. Dr. E. Grill
2. Priv.-Doz. Dr. R.A. Torres Ruiz


Die Dissertation wurde am 28. Okt 2003 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 26. Nov 2003 angenommen.


Contents
Contents

Abbreviations..............................................................................................................................1
Summary ........................................................................................................................................4
General Introduction ..............................................................................................................7
Part I. Signal transduction of abscisic acid in Arabidopsis thaliana…8
1 Introduction..........................................................................................................................9
1.1 ABA signal transduction..........................................................................................9
1.1.1 Identification of ABA signaling intermediates ........................................................10
1.1.2 Protein phosphorylation /dephosphorylation events in ABA signaling...............13
1.1.3 ABA-regulated genes ................................................................................................17
1.2 Homeodomain protein ............................................................................................20
1.2.1 Structures and properties of homeodomain proteins............................................21
1.2.2 Functions of HD-ZIP proteins...................................................................................21
1.2.3 Protein phosphatase ABI1 interacts with Arabidopsis thaliana homeobox
protein AtHB6.............................................................................................................22
1.3 Yeast hybrid system in protein interaction assay ....................................23
1.4 The aim of this work.................................................................................................26
2 Materials and Methods ...............................................................................................29
2.1 Materials.........................................................................................................................29
2.1.1 Microbe materials ......................................................................................................29
2.1.2 Plant material .............................................................................................................29
2.1.3 cDNA libraries............................................................................................................30
2.1.4 Plasmids.....................................................................................................................30
2.1.5 Biochemicals and reagents ......................................................................................34
2.2 Methods..........................................................................................................................35
2.2.1 Cloning techniques ...................................................................................................35
2.2.2 Construction of plasmids .........................................................................................36
2.2.3 DNA analysis techniques..........................................................................................39
2.2.4 Protein analysis .........................................................................................................41
2.2.5 Cellular localization of GUS-fusion proteins ..........................................................48
2.2.6 Two-hybrid system ....................................................................................................49
2.2.7 Three-hybrid yeast system .......................................................................................53
IContents
2.2.8 Agrobacterium-mediated plant cell transformation ...............................................53
2.2.9 Transient expression in protoplasts........................................................................54
3 Results...................................................................................................................................59
3.1 Identification of interaction partners of AtHB6 ..........................................59
3.1.1 Different versions of bait proteins ...........................................................................59
3.1.2 Library screening with the yeast two-hybrid system.............................................61
3.1.3 AtHB7 and AtGluRS ---- the new interaction partners of AtHB6 ...........................63
3.2 Analysis of the interactions between AtHB6, PP2Cs and AtGluRS.73
3.2.1 Two-hybrid interaction of AtHB6 and ABI1/ABI2 ....................................................73
3.2.2 Two-hybrid interaction of AtGluRS and ABI1/ABI2 ................................................75
3.2.3 Physical interaction between ABI1/ abi1 and AtGluRS in vitro.............................78
3.2.4 Co-immunoprecipitation of AtGluRS and ABI1 from maize protoplasts in vivo..83
3.2.5 Ternary interaction among AtHB6, AtGluRS and a third protein in
three-hybrid yeast system........................................................................................93
3.3 Identification of interaction partners of AtGluRS by
a yeast two-hybrid system..................................................................................101
3.3.1 Different versions of bait proteins .........................................................................101
3.3.2 Interaction partners of AtGluRS.............................................................................102
3.3.3 Homodimerization of AtGluRS in yeast.................................................................104
3.4 Analysis of the function of Arabidopsis AtGluRS in yeast ................107
3.4.1 Interactions of the full-length AtGluRS with other proteins in yeast..................107
3.4.2 The potential of Arabidopsis AtGluRS to substitute for yeast AtGluRS ............109
3.5 Effect of AtGluRS on the cellular localization of AtHB6 ...................... 114
3.5.1 Cellular localization of AtGluRS.............................................................................114
3.5.2 AtGluRS alters the cellular localization of AtHB6 ................................................115
3.6 Regulation of Rab18 transcription in transient expression
system by ABI1, AtHB6 and AtGluRS............................................................120
3.6.1 The reporter driven by Rab18 promoter is activated by ABA .............................120
3.6.2 ABI1, AtHB6 and AtGluRS negatively regulate the transcription of Rab18 .......121
3.7 Regulation of the AtHB6-activated transcription in ................................
transient expression system .............................................................................125
3.7.1 Effect of AtHB7 on AtHB6-activated transcription ...............................................127
3.7.2 Suppression of AtHB6-activated transcription by AtGluRS................................128
3.7.3 Stimulation of AtHB6- activated transcription by ABI1........................................133
II Contents
4 Discussion ........................................................................................................................135
4.1 Identification of protein interaction partners
by yeast two-hybrid system screening .................................................135
4.2 Interaction of AtGluRS with AtHB6 and PP2C ..........................................142
4.3 Function of AtGluRS ..............................................................................................146
4.4 A model for the functions of AtGluRS, AtHB6 and
ABI1 in ABA signaling...........................................................................................150
4.5 Does AtHB7 regulate the gene transcription as the AtHB6? ............152
4.6 Is ABI1 conjugated with a small protein in plant.....................................152
Part II. Suppressor screen with an auxin-resi