Characterisation of mutants involved in Phytochrome A nuclear import and signal transduction [Elektronische Ressource] / vorgelegt von Madhusmita Panigrahy

Kish

Sujets

• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 4
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Characterisation of mutants involved in
Phytochrome A nuclear import and signal
transduction



Inauguraldissertation zur Erlangung der Doktorwürde
der biologischen Fakultät
der Albert-Ludwigs-Universität Freiburg im Breisgau






Vorgelegt von
Madhusmita Panigrahy
aus Indien

im October 2004


Dekan: Prof. Dr. Georg Fuchs
Leiter der Arbeit: Prof. Dr. Eberhard Schäfer
Referent: Prof .Dr. Christoph Beck
Koreferent: Prof. Dr. Peter Beyer





Tag der Verkündigung des Prüfungsergebnisses: 07.12.2004









































To my brother, sister-in-law and my nephews and my parents





































Publication related to this study

Bauer D and Panigrahy M, Kircher S, Kunkel T, Adam E, Nagy F and Schäfer E. „Mutants
altered in nuclear-cytoplasmic distribution of the plant photoreceptors phytochrome A and
stphytochrome B.” 1 International SFB 592 symposium. Signalling Mechenism in
Embryogenesis and organogenesis. Signalling Systems: Shaping cells into organs. October 9-
10, 2003. Freiburg. i. Br.

Publication not related to this study

Panigrahy M, Näke C, Harter K, Schäfer K. „Retention of AtCPRF2-2 by LSD1 in Parsley
protoplasts“ Zelluläre Funktionen dynamischer Proteinwechselwirkungen/ SFB 388. May
2001. Freiburg. i. Br.

Näke C, Dittgen J, Panigrahy M, Ellerstorm M, Tang S, Schäfer E, Dangel J, Harter K.
th„LSD1 functions as a retention factor for the bZIP transcription factor AtCPRF2-2.” 13
International Conference on Arabidopsis research. Abstract book page: 6-11. June 28- July 2,
2002. Sevilla, Spain.

























Index
Abbriviations I
Abstract III
1. Introduction
1.1 Role of light in plant development 1
1.2 The Photoreceptors 1
1.2.1 The Cryptochromes and the phototropins 2
1.2.2 The Phytochromes 3
1.3 Molecular characteristics of phytochromes 3
1.4 Structural domains of phytochrome 4
1.5 Mode of action of the phytochromes 5
1.5.1 Low fluence response (LFR) 5
1.5.2 Very low (VLFR) 6
1.5.3 High irradiance (HIR) 6
1.6 Mechenism of phytochrome signal transduction and its components 6
1.6.1 Regulation of expression of light and dark induced genes 7
1.6.2 Phosphorylation 8
1.6.3 Termination of light signal transduction 9
1.6.4 Intracellular partitioning of the photoreceptor 9
1.6.5 Nuclear import and its significance in phytochrome light signal
transduction 11
Table 1: Components and properties of light signal transduction 12
Table 1.1: Nuclear components involved in the signal transduction 12
Table 1.2: Cytoplasmic Com14
Table 1.3: Chloroplast localised components involved in the signal
transduction 14
Table 1.4: Components with unknown subcellular localisation involved
in the signal transduction 14
Table 1.5: Chromophore biosynthesis mutants 15
1.6.6 Perspectives of phytochrome light signalling 15
1.7 Aim of the investigation in this study 16
1.7.1 Introduction of the EMS mutagenesis screen 16
1.7.2 Possible categories of mutants expected from the EMS mutagenesis
screen 16
2. Material and methods
2.1 Seed lines 18
2.2 Standard light fields 18
2.3 Plant work 20
2.3.1 EMS Mutagenesis 20
2.3.2 Screening of the mutants 20
2.3.3 Crossing for genetic analysis 21
2.3.4 Mapping procedure 21
2.3.5 Measurement of hypocotyl growth inhibition 22
2.3.6 Extraction of anthocyanin 22
2.3.7 In vivo spectroscopy 22
2.3.8 Image acquisition and processing 23
2.4 Molecular biology- DNA related techniques 23
2.4.1 CTAB Genomic DNA extraction for mapping and PCR 23
2.4.2 Transformation of chemically competent cells 24

2.4.3 PCR 24
2.4.4 Digestion of genomic DNA for southern blotting 24
2.4.5 Cloning of PHYA: GFP from the mutant 30/83 into TOPO 2.1 25
2.5 Molecular biology- protein related techniques 26
2.5.1 Protein extraction 26
2.5.2 Casting of protein gel for SDS-PAGE 26
2.5.3 Comassie- blue staining 27
2.5.4 Semi-dry western blotting 28
2.5.5 Protein detection and antisera used 28
2.6 Methods followed according to the standard kit protocol 29
2.7 List of Oligonucleotides sequences used in this study 30
2.8 List of PCR markers used for mapping 31
2.9 Web links and software used for data analysis in this study 32
2.10 Chemical list 32
3. Results
3.1 Study of the PSM line 35
3.1.1 Sequencing of Phytochrome A of the PSM line 35
3.2 Characterisation of Transgenic PhyA: GFP (TAG) line 39
3.2.1 Southern blotting analysis 39
3.2.2 Microscopic analysis of phyA: GFP speckle formation in the TAG line 39
3.2.3 Overview of the mutagenesis screening 41
3.3.3 Overview of the mutants obtained 43
3.3.4 Criteria for the mutants selected for further study 44
3.3 Characterization of mutants with a phenotype in darkness
(Figure. 5b: Box 7: 337/85, 240/90) 48
3.3.1 Phenotype of the mutants under FR screening condition and darkness 48
3.3.2 Analysis of phyA: GFP localisation under RG9 screening condition,
strong FR and darkness 48
3.3.3 Effect of 24-Epibrassinolide (EBR) treatment 50
3.4 Characterisation of hyposensitive mutants
(Figure. 5a: Box 1: 361/15, 342/112, 360/15) 52
3.4.1 Phenotype of the mutants under FR screening condition and strong FR 52
3.4.2 Analysis of phyA: GFP localisation under FR screening condition and
strong FR 52
3.4.3 Determination of PHYA: GFP protein amount 54
3.4.3. I. In vivo spectroscopy 54
3.4.3. II. Immunological analysis for detection of PHYA and
PHYA: GFP 54
3.5 Characterisation of hyposensitive mutants
(Figure. 5a: Box 2: 30/83, 381/83, 183/33, 409/102) 58
3.5.1 Phenotype of the mutants under FR screening condition and strong FR
light 58
3.5.2. Fluence response curve analysis 58
3.5.3. Analysis of the phyA: GFP localisation 60
3.5.4. Determination of PHYA amount 62
3.5.4. I. In vivo spectroscopy 62
3.5.4.II. Immunological analysis for detection of PHYA and PHYA: GFP 62
3.5.5 Further characterisation of mutant 30/83 64
3.5.5.1 Genetic analysis 64
3.5.5.2 Characterisation of phyA: GFP in the mutant 66

3.5.5.2.1 Analysis of phyA: GFP localisation in darkness 66
3.5.5.2.2 Further analysis of the phyA: GFP in mutant 30/83 66
3.5.5.3 Analysis of phyA: GFP localisation in different light conditions 69
3.5.5.3.1 Analysis of nuclear speckle formation in the mutant 69
3.5.5.3.2 Analysis of destruction of PHYA: GFP in the mutant 30/83 71
3.5.5.3.3 Altered cytoplasmic speckle formu73
3.5.5.3.4 Microscopic analysis of phyA: GFP speckle formation in the
mutant 30/83 73
3.5.5.3.5 Analysis of phyA: GFP in the mutant in WL 74
3.5.6 Analysis of action spectra of mutant 30/83 and the TAG line 77
3.6 Characterisation of hypersensitive mutants
(Figure 5b: Box 8: 162/16, 334/84, 359/90, 17/57, 358/4, 299/68, 364/90, 155/14,
392/16, 340/90) 803.6.1 Seedling phenotype under the screening condition 80
3.6.2 Fluence rate response curve analysis under FR 80
3.6.3 Further characterisation of weakly hypersensitive mutants under RG9 and
R light 83
3.6.4 Further characterisation of strongly hypersensitive mutants 84
3.7 Characterisation of hypersensitive mutants (Figure 5b: Box 6: 82/8, 338/87) 87
3.7.1 Phenotype of the mutants under FR screening condition and darkness 87
3.7.2 Fluence rate response curve analysis 87
3.7.3 Localisation of phyA: GFP under the RG9 screening condition 89
3.7.4 Further analysis of phyA: GFP localisation 89
3.7.5 Further characterisation of the mutant 82/8 93
3.7.5.1 Determination of phytochrome A amount 93
3.7.5.1.1 Immunological detection analysis 93
3.7.5.1.2 Immunological in situ a