Modes of action of cryptochrome 2 from Arabidopsis thaliana [Elektronische Ressource] / vorgelegt von Rafael Muñoz Viana

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 61
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Modes of action of cryptochrome 2 from Arabidopsis thaliana









DISSERTATION


Zur
Erlangung des Doktorgrades
Der Naturwissenschaften
(Dr. rer. Nat.)









dem Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von




Rafael Muñoz Viana
aus Oviedo/Spanien



Marburg/Lahn im Juni 2007




Die Untersuchungen zur vorliegenden Arbeit wurden von Januar 2004 bis januar 2007 am
pflanzenphysiologie unter der Leitung von Herrn PD Dr. Alfred Batschauer durchgeführt.




















Von Fachbereich Biologie der Philipps-Universitäat Marburg als Dissertation angenommen
am:

Erstgutachter: PD Dr. Alfred Batschauer
Zweitgutachter: Prof Dr. Paul Galland

Tag der mündlichen Prüfung am:
Erklärung


Ich versichere, dass ich meine Dissertation

“Modes of action of cryptochrome 2 from Arabidopsis thaliana“

Selbständig, ohne unerlaubte Hilfe angefertigt und mich dabei keiner anderen als der von mir
ausdrücklich bezeichneten Quellen und Hilfen bedient habe.

Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch keiner anderen
Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient.





Marburg________________ _______________________
(Rafel Muñoz Vian)
















CURRICULUM VITAE

PERSONAL DETAILS:

Name: Rafael Muñoz Viana
Place of birth: Las Palmas de Gran Canaria, Spain
stDate of birth: March 31 , 1977

ACADEMIC DATA:
1992-1995: Bachiller at Auseva High school, Oviedo
1995/2000: First cycle of chemistry at the University of Oviedo.
2000/2002: Second cycle of biochemistry and molecular biology at the University
of Oviedo.
2002: Diploma thesis at the department of biomedical sciences, University
of Antwerp (Belgium) as an Erasmus student (project title: “Effect of
brefeldin A on the cholera toxin induced cAMP accumulation in
Caco2 cells”).
2003-2004: PhD at Helle. D. Ulrich laboratory, Max Plank Institute for Terrestrial
Microbiology Marburg, Germany (thesis title: “Role of PCNA in the
postreplicative DNA repair”.)
2004-2006: PhD at Alfred Batschauer laboratory, department of plant physiology
Philipps university Marburg, Germany (tesis title: “Modes of action
of cryptochrome 2 from Arabidopsis thaliana”).

LANGUAGES:

Spanish: Mother tongue, English: Advanced, German: Basic.

PUBLICATIONS:

Banerjee R., Schleicher E., Meier S., Viana R.M., Pokorny R., Ahmad M., Bittl R.,
Batschauer A. (2007) The signaling state of Arabidopsis cryptochrome 2 contains flavin
semiquinone. Journal of Biological Chemistry 282:14916-14922.

Rosenfeldt G., Viana R.M., Mootz H.D., von Arnim A.G., Batschauer A. (2007)
Chemically induced and light-independent cryptochrome photoreceptor activation. Molecular
Plant (Accepted).

CONGRESSES:

Annual conference of EU RTN group “checkpoint and cancer” Erasmus medical center.
Rotterdam, September 15 2003




Ein Teil der während der Promotion erzielten Ergebnisse wurde in folgender
Originalpublikation veröffentlicht:


Banerjee R., Schleicher E., Meier S., Viana R.M., Pokorny R., Ahmad M., Bittl R.,
Batschauer A. (2007) The signaling state of Arabidopsis cryptochrome 2 contains flavin
semiquinone. Journal of Biological Chemistry 282:14916-14922.

Rosenfeldt G., Viana R.M., Mootz H.D., von Arnim A.G., Batschauer A. (2007)
Chemically induced and light-independent cryptochrome photoreceptor activation. Molecular
Plant (Accepted).



















ACKNOWLEDGEMENTS


First and most of all I would like to express my gratitude to PD Dr. Alfred Batschauer for
trusting and allowing me to work under his supervision, for supporting my research, for his
availability for discussion, for advising my work, and for his never-ending patience during
these past years.

Hence, I would like to thank Oxana Panajotow and Agnes Debelius for their professionalism
and skill-full technical assistance.

Furthermore, I would like to thank each member of the Alfred Batchauer working group for
constructive help, stimulating discussions and good working atmosphere. Special thanks go to
Dr. Richard Pokorny for the great time working together and for his friendship. I would like
also to thank Gebhard A. Kaiser for his technical support.

I am also very grateful to Dr. Israel Fernández López, Carola Rößer, Julia Sommer, Evelyn
Vollmeister Dr. Kenneth Berendzen and Dr. Miroslav Vranes for the help and advice
correcting some chapters of this thesis.

Finally I would also like to thank my family for their constant and no matter-what support
during all these years abroad.














































Dedicated to my family








TABLE OF CONTENTS
TABLE OF CONTENTS

TABLE OF CONTENTS I

LIST OF ABBREVIATIONS V

SUMARY VI

1 INTRODUCTION 1
1.1 Light sensors: photoreceptors 2
1.1.1 Phytochromes (Phys)
1.1.2 Phototropins (Phots) 4
1.1.3 ZTL / LPK2 / FKF1
1.1.4 Cryptochromes (Crys) 5
1.2 Cryptochromes biological role 7
1.2.1 Photomorphogenesis: de-etiolation and gene expression 7
1.2.2 Entrainment of the circadian clock 9
1.2.3 Control of flowering time 11
1.2.4 Biological role of crys in other plants 13
1.3 Cryptochromes structure 13
1.3.1 The cryptochrome N terminus (CNT) 13

1.3.2 The cryptochrome C terminus (CCT) 15
1.4 Crys activation mechanism 17
1.4.1 Cryptochrome Photocycle 17
1.4.2 Early events on cry activation 19
1.4.2.1 Phosphorylation
1.4.2.2 Conformational change. 20
1.4.2.3 Formation of nuclear speckles. 21
1.4.2.4 Degradation and signal turnover. 21

2 MATERIALS & METHODS 23
2.1 Cloning and plasmids 23
2.1.1 General cloning procedure 23
2.1.2 Plasmids 23
I TABLE OF CONTENTS
2.1.3 Cloning Cry2 myc and HA tagged versions 24
2.1.4 Cloning Cry2-GFP, CNT2-GFP and CCT2-GFP into PCV812vector 25

2.2 Plant material, growth conditions, light treatments and bacterial strains 25
2.2.1 Plant material 25
2.2.2 growth conditions 26
2.2.2.1 Growth on soil 26
2.2.2.2 filter paper 26
2.2.2.3 Arabidopsis cel cultre 26
2.2.3 Light treatments 27
2.2.3.1 Light conditions in the growth chambers 27
2.2.3.2 Light treatments for cry phosphorylation 27
2.2.3.3 Light treatments for CO and FT gene expression 27
2.2.3.4 Light treatments for blue light dependent gene expression 28
2.2.4 Agrobacterium strains 28
2.2.4.1 Agrobacterium strains and plasmids used 28
2.2.4.2 growth conditions 28
2.2.4.3 Agrobacterium mediated tobacco infiltration 29
2.3 Protein methods 29
2.3.1 Protein extract preparation 29
2.3.2 measurement by amidoblack 30
2.3.3 Dephosphorylation by Lambda PPase treatment 31
2.3.4 Chemical crosslinking 31
2.3.5 Immunoprecipitation (pull-down) 31
2.3.6 Protein electrophoresis 32
2.3.6.1 SDS-PAGE
2.3.6.2 2D 34
2.3.6.2.1 Protein precipitation 34
2.3.6.2.2 First dimension 35
2.3.6.2.3 Second dime
2.3.6.2.4 Coomassie staining
2.3.7 Western blot 36
2.3.7.1 Blot transfer 36
2.3.7.2 Antibody incubation 36
2.3.7.3 Enhanced chemo-luminiscence development 37
II