Characterization of eid3 and the putative rei mutants [Elektronische Ressource] : new insights on phytochrome A signaling / vorgelegt von Aurora Piñas-Fernandez

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• Biologie

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• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Characterization of eid3 and the putative rei
mutants. New insights on phytochrome A
signaling.



Inagural-Dissertation zur Erlangung der Doktorwürde der
Biologischen Fakultät der Albert-Ludwigs-Universität Freiburg
im Breisgau.



Vorgelegt von Aurora Piñas – Fernandez
aus Spanien

Freiburg, Mai 2003
II
Dekan: Prof. Dr. H. Kleinig
Promotionsvorsitzender Prof. Dr. K. F. Fischbach
Leiter der Arbeit Prof. Dr. E. Schäfer
Referent Prof. Dr. E. Schäfer
Koreferent Prof. Dr. P. Bayer


Tag der Verkündigung des Prüfungsergebnisses: 22 July 2003
III Publications related with this thesis


Piñas A, Dieterle M, Büche C, Schäfer E, Kretsch T. eid3 a new photomorphogenic
Arabidopsis mutant. Conference. October, 2000, Colmar, France.

Piñas che C, Schäfer E, Kretsch T. Study and characterization of eid3 a
new photomorphogenic Arabidopsis mutant. Keystone Symposia “Specificity and Crosstalk
in plant signal transdiction”, Abstract book pp. 63. January 22-27 2002 , Tahoe City, CA,
USA
IV





Als meus pares


V
1.INTRODUCTION ________________________________________________________ 1
1.1. THE PLANTS IN A SURROUNDING LIGHT _________________________________ 2
1.2. THE DISCOVERY OF PHYTOCHROME ____________________________________ 4
1.2.1 Action Spectra Of The High-Energy Reaction __________________________________________5
1.2.2 Phytochrome Is The Receptor For The High- And Low- Energy System______________________7
1.2.3 From The Two-Phytochrome-Pool Theory To The Verification ________8
1.3. PHOTORECEPTORS IN PLANT PHOTOMORPHOGENESIS_________________ 10
1.4. PHYTOCHROME IN Arabidopsis thaliana ___________________________________ 12
1.5. PHYTOCHROME - MEDIATED RESPONSES_______________________________ 14
1.5.1 Role Of Phytochrome - Mediated Responses In Arabidopsis thaliana_______________________15
1.6. PHYTOCHROME SIGNAL AND REGULATION ____________________________ 17
1.7. phyA-SIGNALING MUTANTS _____________________________________________ 20
1.8. AIMS OF THIS WORK ___________________________________________________ 22
2.MATERIAL AND METHODS _____________________________________________ 23
2.1. CHEMICAL LIST ________________________________________________________ 24
2.2. LABORATORY LIGHT ___________________________________________________ 25
2.3. PLANT MATERIAL ______________________________________________________ 29
2.3.1 Wild-Types ____________________________________________________________________29
2.3.2 Mutants_______________________________________________________________________29
2.4. PLANT METHODES AND TECHNIQUES ___________________________________ 29
2.4.1 Methods __________________30
2.4.2 Techniques __________________________________31
2.5. MAPPING TECHNIQUE __________________________________________________ 33
2.5.1 CTAB (Cethyltrimethylammonium Bromide) Plant Genomic DNA Extraction Technique. _______34
2.5.2 Polymerase Chain Reaction _______________________________________________________34
2.5.3 Restriction Digestion And Analysis _________________________________________________35
2.5.4 Mapping Procedure Followed __35
2.5.5 Molecular Markers__________37
2.6. NON-RADIOACTIVE Lhcb 1-NORTHERN BLOT_____________________________ 38
2.6.1 RNA Extraction ______________________________39
2.6.2 Northern Blotting___________________________________________________39
2.6.3 Random Labeling Of DNA With DIG-UTP ________________________39
2.6.4 Hybridization With Dig-Labeled Lhcb 1 ___________________________40
2.6.5 Detection Procedure For Lhcb 1 Dig-Labeled ________________________________________41
2.7. WESTERN BLOT OF PHYOTOCHROME A _________________________________ 41
2.7.1 Protein Extraction And Quantification ______________________________________________41
2.7.2 Electrophoresis Gel _____________________________42
2.7.3 Transfer ______________________________________________________________________43
2.7.4 Detection Of PHYA Protein____43
3.RESULTS _____________________________________________________________ 45
3.1. THE eid3 MUTANT ______________________________________________________ 46
1.1 The eid3 Mutation Maps On Top Of Chromosome Ι ____________________________________46 3.
3.1.2 eid3 Displays Hypersensitivity To Far-Red Light And Dependence On phyA _______________51
3.1.3 The eid3 Mutant Has Not Altered The PHYA Degradation _______________________________54
3.1.4 The eid3 Mutation Confers Hypersensitivity To Continuous Far-Red Light54
3.1.5 eid3 Mutation Confers Hypersensitivity To Very Low Fluence Response____________________61
3.1.6 Comparative Study With Spa1 _____________________________________________________74
VI 3.1.7 Study Of The Eid3 Action Spectra __________________________________________________75
3.2. THE PUTATIVE rei MUTANTS ____________________________________________ 80
3.2.1. Isolation Of The rei- Mutant Lines _______________________________80
3. 2.2.Phenotipic Characterization Of The Putative Rei Lines _________________________________82
4.DISCUSSION __________________________________________________________ 99
4.1. THE eid3 MUTANT ______________________________________________________ 100
4.1.1 EID3 Genes Maps On Chromosome Ι ______________________________________________100
4.1.2. The eid3 Mutant Is Involved On The PhyA-Signaling Pathway __________________________101
4.1.3. Final Notes ________________________________________________106
4.2. THE PUTATIVE rei MUTANTS. ___________________________________________ 108
a) phyA Mutants__________________109
EID1b) Mutants On The Regulatory Pathway Of The SCF Complex ____________________________109
c) Substrate(s) Mutants ____________110
4.3. GENERAL DISCUSION __________________________________________________ 112
APPENDIX_____________________________________________________________ 113
The Ubiquitin-Mediated Degradation Pathway ___________________________________ 113
Photoreceptor mutants________________________________________________________ 114
PhyA signaling related mutants ________________________________________________ 115
PhyB signaling related mutants ________________________________________________ 117
BIBLIOGRAPHY________________________________________________________ 119
Aknowledgments_________ 135
VII Abstract

To identify highly specific components of phytochrome A (phyA) signaling, a new screening program
(SP) was performed. This SP consists of repetitive pulse treatments of 20 minutes red light followed
by 20 minutes far-red light. This multiple pulse program was applied to isolate mutants which were
either impaired in light dependent phyA degradation or exhibited an increased light sensitivity. eid
(empfindlicher im dunkelroten Licht) mutants were isolated from pools of phyB-5 (Ler) seedlings
mutagenised with ethylmethansulfonate (Büche et al., 2000).
eid3 is a dominant mutant which maps on top of chromosome I. To compare the light sensitivity
between eid3 and the wild type, fluence rate response was measured for hypocotyl elongation and
anthocyanin accumulation which are two typical phyA-dependent high irradiance responses (HIR).
Under far-red light conditions both responses were affected. With regard to hypocotyl elongation,
sensitivity was increased by orders of magnitude for VLFR and by one order of magnitiude for HIR.
Anthocyanin accumulation was increased by one order of magnitiude for HIR. Under red light
conditions photoinhibition of hypocotyl growth was hypersensitive to very low fluence rates and the
HIR action spectra was shifted to red light wavelength. In contrast to wild type, eid3 seedlings
accumulated anthocyanin in red light. One explanation to the observed hypersensitivity may be an
overexpression or a increased stability of phyA. However, the eid3 mutant was not altered in the
degradation and levels of phyA. Moreover, phyA is epistatic over eid3 that indicates a strict phyA-
dependency.
The eid1 mutant was isolated under the same screening program as eid3. EID1 encodes an F-box
EID1protein which is a component of an SCF E3 ubiquitin ligase complex. The SCF complex regulates
the phyA signal without impairing the photoreceptor levels (Büche et al., 2000). So far, the substrate
EID1which is recognized by the SCF complex remains unknown. One attempt to identify its substrate
EID1and other components of the SCF pathway is a suppressor screening for the revertants of eid1-3
mutant. The preliminary phenotypical and genetical characterization of the putative rei (revertants of
eid1-3 mutant) lines are presented in this work.

VIII

1.INTRODUCTION 1.1. THE PLANTS IN A SURROUNDING LIGHT
There are clear differences between a plant with flowers and a mammal. One obvious difference is
that a plant is sessile, meaning that it cannot escape adverse surrounding conditions. To solve
surrounding problems, the plant must use physiology whereas the animal can resort to protective
behavior. Studies of plant physiology try to obtain the critical information about the internal
mechanisms taki