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

De
257 pages
Ajouté le : 01 janvier 2003
Lecture(s) : 16
Signaler un abus



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-resistant axr1-12 of.
Arabidopsis thaliana ……………………………………………………………………..…154
1 Introduction......................................................................................................................155
1.1 Fertilization and parthenocarpy .......................................................................155
1.2 Fruit growth is dependent on phytohormones synthesized
in the developing seed..........................................................................................155
1.3 Arabidopsis: an attractive model plant in biology .................................157
1.4 Suppressor screen .................................................................................................160
2 Materials and Methods .............................................................................................161
2.1 Plant materials and growth conditions ........................................................161
...........................................................................................................................1612.2 Screen
2.3 Determination of auxin sensitivity..................................................................162
2.4 Water loss....................................................................................................................162
2.5 Germination ratio.....................................................................................................162
2.6 Apical hook and root gravitropism.................................................................163
2.7 DNA micro-isolation from Arabidopsis leaves:........................................163
2.8 PCR.................................................................................................................................163
2.9 Genetic analysis.......................................................................................................164
3 Results.................................................................................................................................165
3.1 Silique growth of the original axr1-12 mutant and the wild
type plants in response to PGRs.....................................................................165
3.2 Isolation of suppressors from axr1-12 in silique development .......169
3.3 Root growth of suppressors in response to 2,4-D .................................178
3.4 Other physiological characteristics of suppressor lines....................182
3.5 Genetic analysis of the suppressor lines ...................................................185
IIIContents
4 Discussion ........................................................................................................................199
4.1 Plant hormone and fruit development ..........................................................199
4.2 Suppressors of axr1-12 ........................................................................................200
4.3 Gravitropism of suppressor lines ...................................................................202
4.4 Apical hook formation in suppressor lines................................................204
4.5 Seed development in suppressor lines........................................................205
Appendix ....................................................................................................................................206
Appendix 1. Media recipes .............................................................................................206
Appendix 2. Primers...........................................................................................................207
Appendix 3. cDNA and amino acid sequences of the candidates
identified by AtHB6 using yeast two-hybrid system.........................................209
Appendix 4. cDNA and amino acid sequences of the candidates
identified by AtGluRS using yeast two-hybrid system ....................................216
References................................................................................................................................221
References for part I ..........................................................................................................221
References for part II .........................................................................................................236
Acknowledgements ............................................................................................................243
Publications .............................................................................................................................244
Curriculum Vitae ...................................................................................................................245

IV Abbreviations
Abbreviations
aaRS aminoacyl-tRNA synthetase
ABA abscisic acid
ABI1dN N-terminal deleted version of ABI1 (122-434 aa)
ABI1dN262 N-term of ABI1 (262-434 aa)
ABI1NPA N-terminal deleted version of ABI1 (122-434 aa) with one mutation
Asp177Ala and has no phosphatase activity
ABI1Sac C-terminal deleted version of ABI1 (1-268 aa)
ABRE ABA response element
ACC 1-aminocyclopropane-1-carboxylic acid
AD activation domain
Amp ampicillin
AP alkaline phosphatase
APS ammonium persulfate
AtGluRS Arabidopsis glutamyl tRNA synthetase
AtGluRS(1-110) N-terminal version of AtGluRS (1-110 aa)
AtGluRS(1-216) N-terminal version of AtGluRS (1-216 aa)
AtGluRS(1-261) N-terminal version of AtGluRS (1-261 aa)
AtGluRS(-20-261) N-terminal version of AtGluRS (1-261 aa) with additional 20 aa
appended to the N-terminal
AtGluRS(1-445) N-terminal version of AtGluRS (1-445 aa)
AtGluRS(217-445) deleted version of AtGluRS (217-445 aa) 32-45version of AtGluRS (232-455 aa)
AtGluRS(449-719) deleted version of AtGluRS (449-719 aa)
AtHB homeodomain protein in Arabidopsis thaliana
AtHB6dC119 C-terminal deleted version of AtHB6 (1-119 aa)
AtHB6dC217 inal deversion of AtHB6 (1-217
AtHB6dC269 inal deleted version of (1-269 aa)
AtHB7dC C-terminal deleted version of AtHB7 (1-136 aa) with additional 16 aa
appended to the N-terminal
Ath Arabidospsis thaliana
AXR auxin resistance gene
ATP adenosine 5’-triphosphate
BAP benzyladenine
BCIP 5-bromo-4-chloro-3-indolyphosphate
bp base pair
BSA bovine serum albumin
CaMV Cauliflower mosaic virus
cDNA complementary deoxyribonucleic acid
CE coupling element
cfu colony forming units
CIP calf intestine phosphatase, alkaline
1Abbreviations
CTAB cetyltrimethylammoniumbromid
2,4-D 2,4-Dichlorophenoxyacetic acid
DAPI 4’,6’-diamidino-2-phenyl-indol-dihydrochlorid
DAS days after sowing
DBD DNA binding domain
DMF Dimethylformamid
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DTT dithiotreitol
EDTA ethylenediamine tetraacetic acid
EMS methanesulfonic acid ethyl ester
FDA fluorescein diacetat
GAs gibberellins
GAS AtGluRS-antisense line
GluRS glutamyl tRNA synthetase
GS AtGluRS-sense line
GST glutathione S-transferase
GUS β-D-glucuronidase
GuSCN Guanidinthiocyanat
HA hemagglutinin
HD homeodomain
HRP horseradish peroxidase
IPTG isopropyl β-D-thiogalactoside
Kan kanamycin
kb kilobase
kDa kilo Dalton
Lu light units
LUC luciferase
MES 2-morpholinoethansulfone acid
MetRS methionyl-tRNA synthetase
MUG 4-methylumbelliferon-β-D-glucuronid
NBT nitroblue tetrazolium
NLS nuclear localization signals (sequences)
NOS napaline synthase
ONPG O-nitrophenyl β-D-galactopyranoside
ORF open reading frame
Ori origin of replication
PAGE polyacrylamide gel electrophoresis
PBS phosphate-buffered saline
PCR polymerase chain reaction
PEG polyethylene glycol
PGRs plant growth regulators
2

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