Identification of growth-related tonoplast proteins in Arabidopsis thaliana [Elektronische Ressource] / von Samuel Janne Arvidsson

Samuel Janne Arvidsson

Sujets

• Biologie

Informations

• Publié par: universitat_potsdam
• Publié le: 01 janvier 2010
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Institut für Biochemie und Biologie
Arbeitsgruppe Prof. Dr. Bernd Müller-Röber


Identification of
growth-related tonoplast proteins
in Arabidopsis thaliana


Dissertation
zur Erlangung des akademischen Grades
„doctor rerum naturalium“
(Dr. rer. nat.)

in der Wissenschaftsdisziplin „Molekularbiologie“

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät
der Universität Potsdam

von

Samuel Janne Arvidsson
geboren am 15.12.1979 in Örkelljunga, Schweden

Potsdam, Oktober 2010
 













































Published online at the
Institutional Repository of the University of Potsdam:
URL http://opus.kobv.de/ubp/volltexte/2011/5240/
URN urn:nbn:de:kobv:517-opus-52408
http://nbn-resolving.org/urn:nbn:de:kobv:517-opus-52408 Declaration
 
Declaration

I hereby declare that this Ph.D. thesis is the result of my own work carried out
between February 2007 and September 2010 in the group of Prof. Dr. Bernd Müller-
Röber at the University of Potsdam in Potsdam-Golm, Germany, written by me alone
using solely the cited sources and described methods. Additionally I declare that I
have not until now tried to submit or defend a Ph.D. thesis, neither at the University of
Potsdam nor at any other university.


Potsdam, 08.10.2010


Samuel Arvidsson

i  
ii Acknowledgements
Acknowledgements

First of all, I would like to express my gratitude to Prof. Dr. Bernd Müller-Röber, who
made it possible for me to come to do my Ph.D. work in his lab, and for his motivation
and supervision during the years here. Also, thanks to Dr. Katrin Czempinski, I was
able to take part in the VaTEP project, which she organized with excellence; thanks
to this project I have had many possibilities to interact with and visit other research
groups as well go to many interesting conferences. Also, I’d like to thank the
reviewers of this thesis for taking the time and effort.
Another big thanks goes to the greenhouse personnel, especially Christiane Schmidt
and Doreen Mäker, for taking good care of the plants and offering a lot of help for my
work. I’d also like to thank the Greenteam at the MPI, mainly Karin Köhl and Linda
Bartetzko for their help with the plants I grew there. Also, without the work of
Sebastian Kopp, Franziska Jeschke, Paul Saffert and Elisa Schulz, who helped out
with genotyping and various other lab tasks, I wouldn’t have managed to obtain all
the data presented in this thesis.
Thanks to the EU-funded project VaTEP (‘Vacuolar transport equipment for growth
regulation in plants’, MRTN-CT-2006-035833) I had funding for the first three years of
Ph. D. studies, and thanks to the BMBF-funded project GoFORSYS (‘Potsdam-Golm
BMBF Forschungseinrichtung zur Systembiologie. Photosynthesis and Growth; a
Systems Biology Based Approach’, FKZ 0313924), I had time to finish up the
experiments and thesis writeup.
I would also like to thank Prof. Dr. Diego Mauricio Riaño-Pachón, Dr. Miros ław
Kwa śniewski, Dr. Paulino Pérez-Rodríguez, Flavia Vischi Winck, Magda Łotkowska
and Muhammad Arif for the nice collaborations and contributions to the work
presented in this thesis, and all other current and former Mü-Rös, especially Dr. Luiz
Gustavo Guedes Corrêa and Dr. Fernando Alberto Arana Ceballos for the nice time
in and outside of the lab.
Last, but certainly not least, I would like to thank my family for their support during
these and former years and especially my wife Sandra, for her continuous love,
support and understanding for the long working days, the late nights and weekends
in front of the computer writing up this thesis.
Also, I would like to thank all others who have helped me and were not mentioned
here.
iii  


iv Table of contents
 
Table of contents

Chapter Title Page
Declaration i
Acknowledgements iii
Table of contents v
List of figures vii
List of tables viii
List of abbreviations ix
Summary xi
1 General introduction 1
1.1 Introduction to plant vacuoles 1
1.2 Diversity of plant vacuoles and their different roles 3
1.3 The tonoplast and its constituents 4
1.4 The role of the vacuole and the tonoplast in growth 5
1.5 Leaf cell elongation and leaf organ size control 6
1.6 Expression analysis with real time RT-qPCR 7
1.7 Leaf growth phenotyping 12
1.8 Aims and structure of the thesis 15
2 High-throughput RT-qPCR primer design 17
2.1 Authors’ contributions 17
2.2 Abstract 18
2.3 Background 18
2.4 Implementation 19
2.5 Results 28
2.6 Discussion 29
2.7 Conclusion 29
2.8 Methods 29
2.9 Availability and requirements 31
2.10 Authors' contributions 31
2.11 Acknowledgements 31
2.12 References 31
3 A growth phenotyping pipeline for Arabidopsis thaliana 33
3.1 Summary 33
v Table of contents
 
3.2 Introduction 33
3.3 Results 35
3.4 Discussion 50
3.5 Materials and methods 53
4 Identification of leaf growth-related tonoplast protein genes 57
4.1 Summary 57
4.2 Introduction 57
4.3 Results 58
4.4 Discussion 69
4.5 Materials and methods 72
5 General discussion and outlook 79
5.1 Overview with summary 79
5.2 QuantPrime – a tool for improved RT-qPCR platform design 79
5.3 Plant growth phenotyping 81
5.4 Identification of growth-related tonoplast protein genes in A. thaliana 84
6 References 87
Supplementary material 97
Allgemeinverständliche Zusammenfassung 147
List of publications 149
Curriculum vitae 151
vi List of figures
 

Figure Title Page
1.1 A schematic representation of the plant cell. 1
2.1 'Primer finding' in QuantPrime. 21
2.2 'Results' in QuantPrime. 22
2.3 'Primer pair details' in QuantPrime. 23
2.4 Overall work flow of primer pair design and specificity testing. 24
2.5 Work flow overview of the primer pair design algorithm. 25
2.6 Work flow overview of the primer pair specificity testing algorithm. 26
3.1 The imaging chamber. 36
3.2 Example of a plant image before and after processing. 37
3.3 Plot of CV of technical replicate measurements against mean 39
rosette area.
3.4 Screenshots of the annotation tool. 40
3.5 Smoothed fits for three phenotypic parameters using four different 41
predictor variables.
3.6 Linear mixed-effects model fit for rosette area and RGR of sex4-3. 44
3.7 Development times box plots. 46
3.8 Development stage plots for phenotypes. 48
3.9 Linear mixed-effects model grf9. 50
4.1 Schematic of leaf samples used for expression analysis. 61
4.2 Area and RGR modeling for nhx4 mutants. 66
4.3 exp3, exp6, grf9 and vha-e3 mutants. 66
4.4 Photos of representative mutant plants at 14 DASE. 67
4.5 Plant development comparison for nhx4 mutants. 68

vii List of tables
 
List of tables

Table Title Page
2.1 Examples of transcriptome annotations available on the public 20
QuantPrime server
2.2 Results of in silico benchmarking of QuantPrime 27
2.3 Experimental results of primer pairs designed with QuantPrime 28
3.1 Comparison of phenotyping capabilities and plant densities for 37
different tray types.
3.2 Comparison of observed phenotype values for sex4-3 and WT 45
between five experiments.
3.3 Development times for sex4-3 and WT. 47
3.4 Rosette areas of sex4-3 and WT at different growth stages. 48
4.1 A summary of the RT-qPCR platform. 60
4.2 Scoring template for growth-association of genes. 61
4.3 The genes with the highest scores for growth-association. 62
4.4 Knockout lines for which homozygous progeny was obtained and 64
which were screened with growth genotyping.
4.5 Growth effects for genotypes showing significant effects when 65
modeling area over time.
4.6 Significant plant development effects for genotypes showing 67
significant effects – early developmental stages.
4.7 Significant plant development effects for genotypes showing 68
significant effects – late developmental stages.

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