Functional analyses of stress-induced genes with essential roles in abiotic stress adaptation in the moss Physcomitrella patens (Hedw.) Bruch & Schimp [Elektronische Ressource] / by Enas A. Qudeimat
139 Pages
English

Functional analyses of stress-induced genes with essential roles in abiotic stress adaptation in the moss Physcomitrella patens (Hedw.) Bruch & Schimp [Elektronische Ressource] / by Enas A. Qudeimat

-

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer

Informations

Published by
Published 01 January 2009
Reads 29
Language English
Document size 14 MB



Functional Analyses of Stress-
Induced Genes with Essential Roles
in Abiotic Stress Adaptation in the
Moss Physcomitrella patens (Hedw.)
Bruch & Schimp






By

Enas A. Qudeimat
Born in Kuwait




Inaugural-Dissertation zur Erlangung der Doktorwürde
der Fakultat fur Biologie der Albert-Ludwigs-
Universität Freiburg im Breisgau





June 2009


Current Dean: Prof. Dr. Ad Aertsen

Thesis Advisory Committee:
Ph.D. Chairman: Prof. Dr. Eberhard Schäfer
Supervisors: Prof. Dr. Ralf Reski & PD Dr. Wolfgang Frank
Referent: PD Dr. Thomas Kretsch
Korreferent: Prof. Dr. Ralf Reski

Day doctorate awarded: 28.July 2009




















This work has been done in the
Institute of Biology II,
Department of Plant Biotechnology
Albert-Ludwigs-Universität Freiburg im Breisgau
under the supervision of Prof. Dr. Ralf Reski and PD Dr. Wolfgang Frank

ii
TABLE OF CONTENTS
ACKNOWLEDGMENTS ......................................................................................................................v
LIST OF PUBLICATIONS AND MANUSCRIPTS ............................................................................... vi
ADDITIONAL CONTRIBUTIONS ...................................................................................................... vii
ABSTRACT ........................................................................................................................................ ix
1 INTRODUCTION...........................................................................................................................1
1.1 The Moss Physcomitrella patens.........................................................................................1
1.2 Abiotic Stress Signal Transduction in Plants .......................................................................3
1.2.1 Salt Stress...............................................................................................................3
1.2.2 Dehydration .............................................................................................................4
1.2.3 Cold Stress..............................................................................................................5
1.2.4 Role of ABA in Abiotic Stress ..................................................................................6
1.3 Calcium as a Secondary Messenger in Signal Transduction...............................................7
1.4 Tetrapyrrole Biosynthesis and Translocator Proteins ..........................................................8
2 AIMS OF THE THESIS................................................................................................................11
3 RESULTS AND DISCUSSION ....................................................................................................13
3.1 Characterization of PCA1: a Vacuolar Calcium Pump in Physcomitrella...........................13
3.2 Characterization of TSPO1: A Key Regulator of the Tetrapyrrole Biosynthesis Pathway in
Physcomitrella ...................................................................................................................16
3.2.1 The Physcomitrella Genome Encodes Five TSPO Proteins..................................20
3.2.2 TSPO Proteins are Transmembrane Proteins .......................................................23
3.2.3 Localization of TSPO Proteins...............................................................................24
3.2.4 Regulation of TSPO Genes in Physcomitrella27
3.3 Role of ABA in Abiotic Stress Responses in P. patens......................................................29
4 REMARKS AND OUTLOOK........................................................................................................30
5 REFERENCES............................................................................................................................32
6 PUBLICATION I...........................................................................................................................39
7 ION II........................................................................................................................ 55
8 PUBLICATION III.........................................................................................................................59
9 MANUSCRIPT I...........................................................................................................................79
10 SUPPLEMENTARY DATA AND METHODS.............................................................................118
10.1 Available Data for TSPO Proteins of Physcomitrella in the COSMOSS Database ..........118
10.2 Exon-Intron Structure of Putative TSPO Genes ..............................................................118
10.3 Prediction of Transmembrane Domain ............................................................................119
10.4 Cloning of the TSPO::GFP-Fusion Constructs of Physcomitrella and Arabidopsis..........120
10.5 Localization of AtTSPO Protein .......................................................................................122
10.6 Targeted Knockout of PpTSPO2 & 3...............................................................................123
10.7 Functional Analysis of TSPO Genes in Arabidopsis ........................................................124
10.8 Accession Numbers of Mutants Used For the Functional Analysis..................................125
GLOSSARY.....................................................................................................................................126
DECLARATION ...............................................................................................................................128
iii
iv
ACKNOWLEDGMENTS

I am grateful to Prof. Dr. Ralf Reski for giving me the opportunity to work in his group, for his
support, suggestions and trustful help in so many aspects.

Especially, I need to express my gratitude and deep appreciation to PD Dr. Wolfgang Frank for all
the effort he has put into me and my work during the time it took me to complete my Ph. D. For your
full supervision, help and your patience: Danke vielmals.

I am grateful to many people who shared their experiences and knowledge; especially Richard Haas
for his technical support, and my practical student Verena Adolf for her hard work and eagerness to
learn. For old and new members of AG Wolle; Kim-Miriam Baar, for taking the time to teach me in
the beginning of my molecular knowledge. Dr. Sandra Richardt, for being there to answer my
questions concerning cosmoss database and discuss how to make a better thesis. Dr. Gotelinde
Seumel, for your support and for always being there. Muhammad Asif Arif, for helping me by making
successful cloning strategies. Philipp Rödel, Marta Tomek and Isam Fattash for your
encouragement and concern.

Thanks also extend to all the other members of AG Reski for the friendly and cooperative work
atmosphere and for sharing lots of unforgettable memories during the last four years. I would also
like to acknowledge the diligent work of the bioinformatics team represented by PD Dr. Stefan
Rensing, Dr. Daniel Lang and Andreas Zimmer.

I am grateful too for the support and advice I have got during my stay in the UK from Prof. Dr. Colin
Brownlee and Dr. Glen Wheeler regarding calcium measurements. To you Jain Bennett for your
hospitality and for making nice tea just at the right moment.

For you Anne Katrin Prowse, Dr. Gotelinde Seumel and Dr. Muawia Qudeimat for proofreading my
thesis.

I am in debt to the Deutscher Akademischer Austausch Dienst (DAAD) for their generous
sponsorship which made it possible for me to pursue my Ph. D. degree 3000 km away from home. I
would also like to thank my referent Mrs. Birgit Klaes for her good-natured support and assistance
throughout my scholarship

Last but not least, thanks to all my family members for all the support I always received, especially
my parents; Amneh Al-Hroub and Abdullah Qudeimat; without your devoted help I would not be
here today. For my partner and best colleague, Basel Khraiwesh, for being open minded, supporting
and always offering me a loving and challenging environment to pursue my goals.
v
LIST OF PUBLICATIONS AND MANUSCRIPTS

This thesis is based on the following publications and manuscripts; they are cited as indicated
below:

Publication I
Qudeimat E., Faltusz A., Wheeler G., Lang D., Brownlee C., Reski R., Frank W. (2008) A P -type IIB
2+Ca -ATPase controls abiotic stress signaling in moss. Proceedings of the National Academy of
Sciences of the United States of America (PNAS). 105: 19555-19560.
Own contribution:
Analyzing expression of the PCA1 gene in the moss Physcomitrella patens in response to abiotic
stresses, studying the expression of two stress-induced genes in both wild type and knockout
plants, conducting all phenotypic screening experiments, carrying out all calcium measurements,
related topics and identification of single insertion event of the PCA1 knockout lines by genomic
Southern blot. Additionally, processing data, performing statistical analysis and generation of the
figures related to the performed experiments and writing the experimental procedure of the related
parts.

Publication II
2+ 2+Qudeimat E. and Frank W. (2009) Ca signatures: The role of Ca -ATPases. Plant Signaling &
Behavior. 4: 350 - 352.
Own contribution:
Writing of the manuscript and modeling the calcium ions as a signaling molecule upon stress
treatments in Physcomitrella.

Publication III
Frank W., Baar KM., Qudeimat E., Woriedh M., Alawady A., Ratnadewi D., Gremillon L., Grimm B.,
Reski R. (2007) A mitochondrial protein homologous to the mammalian peripheral-type
benzodiazepine receptor is essential for stress adaptation in plants. Plant J 51: 1004-1018.
Own contribution:
Expression analysis of TSPO1 in wild type Physcomitrella at early time points upon abiotic stress
treatments, identification of single insertion event of the TSPO1 knockout lines by genomic Southern
blot, analyzing the phenotype of the knockout lines i.e. growing knockout lines in the dark upon salt
treatment and treatment with the diphenyl-ether herbicide acifluorfen, measurements of ALA
synthesis rates, sample preparation to measure protoporphyrin IX concentrations and finally
processing data, statistical analysis and generation of figures related to the performed
experiments.…


vi B

Manuscript I
Richardt S., Timmerhaus G., Lang D., Qudeimat E., Reski R., Rensing SA., Frank W. (2009)
Microarray analysis of the moss Physcomitrella reveals evolutionarily conserved transcriptional
regulation of salt stress and abscisic acid signaling.
Manuscript submitted to Plant Molecular Biology in revision.
Own contribution:
Identification of 9-cis epoxycarotenoid dioxygenase (NCED) gene family in Physcomitrella, key
regulators of abscisic acid (ABA) biosynthesis in plants and validation of their expression upon
different stress treatments. Validation of novel Physcomitrella salt/or ABA responsive TAP genes
(PpABI3B; AP2/EREBP and PpACP1) by semi-quantitative RT-PCR and RNA gel blots.


ADDITIONAL CONTRIBUTIONS

Oral Presentations
- Qudeimat E., Reski R. and Frank W. (2008) Signaling and adaptation: essential genes
underlying abiotic stress tolerance in Physcomitrella patens. The Annual International
Symposium for Moss Experimental Research (Moss 2008), August 15–18, Tampere,
Finland. This talk was presented by: PD Dr. Wolfgang Frank.

2+
− Qudeimat E., Faltusz A., Seumel G., Brownlee C., Reski R., Frank W. (2007) A P -type Ca -IIB
ATPase controls abiotic stress signaling in moss. Botanical Congress (Botanikertagung),
September 3-7, University of Hamburg, Germany.

Poster
- Qudeimat E., Adolf V., Reski R. and Frank W. (2008) Identification of homologues of the
mammalian peripheral-type benzodiazepine receptor in Physcomitrella patens. The Annual
International Symposium for Moss Experimental Research (Moss 2008), August 15–18,
Tampere, Finland.










vii
viii