Functional and molecular characterization of STH1 [Elektronische Ressource] : similarities and differences to the homolog STO / presented by Felipe Sarmiento Salazar

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• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Functional and Molecular
Characterization of STH1: Similarities
and Differences to the Homolog STO

Inaugural-Dissertation
To obtain the Doctoral Degree
Faculty of Biology
Albert-Ludwigs-Universität
Freiburg im Breisgau

Presented by
Felipe Sarmiento Salazar
From Bogotá, Colombia













December 2010









Dean of the Faculty of Biology: Prof. Dr. G. Neuhaus
Chairman of the committee for doctoral studies: Prof. Dr. S. Rossel
Referent: Prof. Dr. G. Neuhaus
Co-referent: PD. E. Decker


Day of the presentation of the thesis: 20.12.2010



The present work was done from April 2006 to October 2010 under the direction
of Prof. Dr. Gunther Neuhaus, Institute of Biology II, Department of Cell Biology________________________________________________________________________
Table of Contents

Abbreviations ..................................................................... iv
1. Introduction ..................................................................... 1
1.1. Photomorphogenesis in plants ................................................................... 1
1.1.1. Blue light photoreceptors ..................................................................... 1
1.1.2. Red and far-red light photoreceptors ................................................... 3
1.1.3. Signalling networks during photomorphogenesis ................................. 5
1.2. The BBX protein family ............................................................................... 9
1.3. Regulation of the circadian rhythm in plants ............................................. 11
1.4. Light and circadian control of the cold stress responses .......................... 12
1.5. RCD1 has multiple roles in hormone and abiotic stress processes .......... 14
1.6. Aim of the work ......................................................................................... 16
2. Material and methods ................................................... 17
2.1. Material .................................................................................................... 17
2.1.1. Chemicals .......................................................................................... 17
2.1.2. Instrumentation .................................................................................. 17
2.1.3. Enzymes and molecular biology kits .................................................. 17
2.1.4. General buffers and solutions ............................................................ 18
2.1.5. Culture media .................................................................................... 19
2.1.6. Nucleic acids ...................................................................................... 20
2.1.7. Bacterial strains ................................................................................. 24
2.1.8. Yeast strains ...................................................................................... 24
2.1.9. Plant material ..................................................................................... 24
2.2 Methods .................................................................................................... 26
2.2.1. DNA-Modification and quantification of nucleic acids ........................ 26
2.2.2. Polymerase chain reaction (PCR) ...................................................... 26
2.2.3. DNA isolation and separation ............................................................ 27
2.2.4. Extraction of total RNA from plants .................................................... 28
2.2.5. Electrophoresis of RNA in denaturing gels ........................................ 29
2.2.6. Northern blotting ................................................................................ 29
i________________________________________________________________________
2.2.7. Quantitative Real Time PCR using SYBR® Green ............................ 31
2.2.8. Bacterial methods .............................................................................. 31
2.2.9. Yeast methods ................................................................................... 32
2.2.10. Plant .................................................................................. 33
2.2.11. Phenotypic analyses ........................................................................ 34
2.2.14 Cross-fertilization of Arabidopsis plants ............................................ 36
2.2.15 Database addresses and Software used .......................................... 36
3. Results ........................................................................... 38
3.1. Common features in the regulation and function of STO and STH1......... 38
3.1.1. STH1 transcription is regulated by the circadian clock ...................... 38
3.1.2. Salt stress experiments with STH1-expressing yeast ........................ 39
3.2. Characterization of STH1 gain- and loss-of-function lines ........................ 42
3.2.1. Phenotypic analyses of STH1 gain- and loss-of-function lines in light
..................................................................................................................... 45
3.3. Genetic interactions of STH1 with photomorphogenic mutants ................ 50
3.3.1. STO – STH1 interplay in plants ......................................................... 50
3.3.2. Genetic interaction of CRY1, PHYA and HY5 with STH1 .................. 53
3.4. Light, circadian and cold regulation of STO and STH1 transcripts ........... 54
3.4.1. Analysis of CAB3 circadian regulation in STO and STH1 loss-of-
function lines ................................................................................................ 54
3.4.2. Circadian rhythm analyses of STH1 and STO in circadian clock
mutants ........................................................................................................ 55
3.4.3. Expression analyses of STH1 and STO in cbf2-2 and CBF/DREB1
overexpression lines .................................................................................... 60
3.4.4. Influence of light and circadian regulation in the transcriptional
response of STO and STH1 to cold stress .................................................. 63
3.5. Interplay between STO and RADICAL INDUCED CELL DEATH1 (RCD1)
........................................................................................................................ 75
3.5.1. Phenotypic analyses of rcd1-3 and sro1-1 loss-of-function mutants
under monochromatic light .......................................................................... 75
3.5.2. Genetic interaction of STO and RCD1 during photomorphogenesis .. 77
ii________________________________________________________________________
3.5.3. Genetic interaction of STO and SRO1 during photomorphogenesis .. 79
3.5.4. Genetic interaction between STO and RCD1 in oxidative stress ....... 80
3.5.5. Expression analyses of STO under paraquat stress. ......................... 82
4. Discussion ..................................................................... 85
4.1. Similarities and differences in the function and regulation of STO and
STH1 ............................................................................................................... 85
4.2. Regulation of the transcription of STO and STH1 by the circadian clock . 89
4.3. Cold regulation of the transcription of STO and STH1 ............................. 89
4.4. Genetic interaction between STO – RCD1 ............................................... 94 cSTO and SRO1 ........................................... 97
5. Summary ........................................................................ 99
6. References ................................................................... 100
7. Supplementary Data .................................................... 118
7.1. Inverse PCR results ............................................................................... 118
7.2. Phenotypic characterization of 35S:STH1 overexpression lines ............ 119
7.3. Characterization of 35S:STH1:GFP-L69/sto-1 lines ............................... 120
7.4. Characterization of the T-DNA lines ....................................................... 121
7.4.1. Characterization of the cbf2-2 T-DNA line ....................................... 121
7.4.2. Characterization of the toc1-3 line ................................................... 122
7.4.3. Characterization of the lhy-31 line 123
7.4.4. Characterization of gi-12 .................................................................. 126
8. Acknowledgements ..................................................... 128

iii________________________________________________________________________
Abbreviations
aa amino acid(s)
ABA abscisic acid
32 32
α- P-dCTP dCTP radioactive labelled with isotope Phosphate at α position
Amp ampicillin
APX1 ascorbate peroxidase1
ATP adenosine 5´-triphosphate
Bc continuous blue light
bHLH basic helix loop helix
bp base pairs
bZIP basic region/leucine zipper motif
°C degrees Celsius
2+Ca calcium ion(s)
CAB3 chlorophy