Transcriptional regulation of root stem cells
by WOX5 in Arabidopsis thaliana
Inaugural-Dissertation
Zur Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau
vorgelegt von
Thomas Friedrich
aus Herrenberg, Deutschland
Dezember 2015
Table of Contents
Summary.................. .................................................................................................... ................ 9
Introduction.........................................................................
11
The shoot apical meristem and the WOX gene family................................... ................ 13
The root apical meristem.......................................................................................................15
The role of WOX5 in root stem cell regulation......................................................... ........ 17
Transcriptome analysis of root cells with altered W 0X5 levels..................................... 19
Evolution of WOX gene function............ ............................................................................. 20
Aims of the study......................................................................................................................... 25
Results....................................................................................................................
26
Chapter I: Transcriptional regulation of root stem cells by WOX5 .................................... 26
1.
Functional analysis of HANABA TARANU...................................................................26
1.1 Transcriptome data and working hypothesis for HAN ........................................... 26
1.2 Is HAN expression in the root regulated by WOX57 .............................................28
1.3 Does WOX5 directly bind to the HAN promoter to regulate its expression?.....29
1.4 The role of HAN in the distal root meristem........................................... ........ ....... 33
1.5 The role of HAN and GIF1 in the proximal root meristem.....................................35
1.6 Ectopic HAN expression in the root tip promotes CSC fate.................. ....... ...... 36
1.7 Does HAN act downstream of WOX5 in CSC regulation?...................... ............. 38
1.8 Possible functions of HAN in the regulation of CSCs downstream of WOX5.... 43
1.9 Conclusions for H A N .........................................................
47
2.
Functional analysis of GROWTH REGULATING
FACTORS (GRFs),
GRF
INTERACTING FACTRORS (GIFs) and microRNA396 (miR396)................................48
2.1 Transcriptome data and working hypothesis for GRFs and G IFs ........................48
2.2 The role of GRFs and GIFs in the distal root meristem.......................................... 50
2.3 Expression pattern of GRFs in the root tip....................................
52
2.4 Expression pattern of miR396.....................................................................................55
2.5 Expression pattern of GIFs in the root tip .................................................................57
2.6 Do GIFs and GRFs act downstream of WOX5 in stem cell regulation?..............60
2.7 What is the role of miR396 in the distal root meristem?.........................................62
2.8 Conclusions for GRFs, GIFs and miR396 .................................................................65
3.
Functional analysis of CLE 11,12 and 13 ...................................................................67
3.1 Microarray results and working hypothesis for CLE11-13 .................................... 67
3.2 Expression pattern of CLE13 ...................................................................................... 69
3.3 Roles of CLE11-13 in the distal root meristem........................................................70
3.4 Conclusions for CLE11-13 ........................................................................................... 72
4.
Functional analysis of W O X 7 .......................................................
74
4.1 Microarray observations and working hypotheses........ ........................................ 74
4.2 Is WOX7 repressed in QC cells by WOX5? ............................................................75
4.3 Is WOX7 upregulated in wox5-1 QC cells to compensate for the loss of WOX5?
78
4.4 Conclusions for W O X 7 ....................................................
5.
79
Functional analysis of PISTILLATA (P I) ...................................................................... 80
5.1 Transcriptional changes of PI in microarray data and working hypothesis.......80
5.2 Does upregulation of PISTILLATA contribute to the wox5 mutant phenotype? 81
5.3 Conclusions for PISTILLATA ................................................
83
Chapter II: Arabidopsis WOX13 and WOX14 are not involved in root meristem
generation.................................................................................................................................... 84
Discussion.................................................................................................................................... 87
How does WOX5 regulate columella stem cells?......................... ............................... 87
How does WOX5 influence QC identity?........................................................................ 89
Molecular mechanism of gene regulation by WOX5 ..................................................... 90
Which target candidate genes act in the root meristem independently of W OX57 .91
Why did we find many target gene candidates that could not be confirmed?......... 92
What is the molecular function of HAN downstream of W OX57 ................................93
WOX13-like genes do not contribute to root tip regeneration in Arabidopsis.......... 94
What is the role of WOX13-like genes in Arabidopsis thaliana? ................................95
6.6 Future work.................................................................................................................... 95
Materials and Methods..............................................................................................
98
Deposition of data and materials generated during this thesis................................... 98
Chemicals and Enzymes....................
98
Plant material............................................................
98
Mutants.............................................................................................
98
Marker lines..........................................................................................................................99
Transgenes used in this study.............................................................
100
Methods................ ................................................................................................................ 103
Flourescence activated cell sorting (FACS).......................................................
103
Microarray data analysis and selection of candidate genes......... .............................103
Plant W ork...............................................................
103
Plant Transformation
104
Selection of Transgenic Plants........................................................................................104
GUS staining........................................................................................................................104
Nomarski Microscopy........................................................................................................105
Microscopy of fluorescent marker lines.......................................................................... 105
Dexamethasone treatments............................................................................................. 105
Preparation of DMA from plant material......................................................................... 106
Primers used for genotyping in this study:.................................................................... 107
Genotyping by P C R s .........................................................................................................107
Standard PCR conditions.................................................................................................108
Root regeneration assay................................................................................................... 108
Cloning of transgenes........................................................................................................108
Ligation Independent Cloning.......................................................................................... 109
References..................................................................................................................................111
Abbreviations............................................................................................................................. 118
Appendices.................................................................................................................................120
Appendix 1: Primers used for cloning of Transgenes:.................................................... 120
Appendix 2: Microarray results............................................................................................ 127
Acknowledgements................................................................................................................... 153