ebook img

Regulation of the floral transition at the shoot apical meristem of Arabidopsis as studied by genetics PDF

224 Pages·2010·5.58 MB·English
by  
Save to my drive
Quick download
Download
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.

Preview Regulation of the floral transition at the shoot apical meristem of Arabidopsis as studied by genetics

Regulation of the floral transition at the shoot apical meristem of Arabidopsis as studied by genetics and next generation sequencing Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Stefano Torti aus Narni (Italien) Köln, April 2010 Die vorliegende Arbeit wurde am Max-Planck-Institut für Pflanzen Züchtungsforschung in Köln, in der Abteilung für Entwicklungsbiologie der Pflanzen (Direktor Prof. Dr. George Coupland) angefertigt. Prüfungsvorsitzender: Prof. Dr. Martin Hülskamp Berichterstatter: Prof. Dr. George Coupland Prof. Dr. Wolfgang Werr Tag der mündlichen Prüfung: 9. Juni 2010 Abstract The floral transition is the process by which flowering plants switch from vegetative growth to the production of flowers. Consistent with the importance of this developmental transition, flowering is highly regulated through several genetic pathways, some of which respond to environmental cues. Arabidopsis thaliana flowers earlier under long-days (LD) of spring than under short-days (SD) of winter, and day-length, or photoperiod, is one of the most important environmental stimuli influencing the flowering response. Photoperiod is perceived in the leaves, while the floral transition occurs at the shoot apical meristem (SAM). In LD, a genetic cascade is activated in the leaf vasculature, so that a key transcriptional regulator called CONSTANS activates the genes FLOWERING LOCUS T (FT) and its homolog TWIN SISTER OF FT (TSF). FT protein is then transported through the phloem, eventually reaching the SAM, where it triggers the floral transition. By forming a complex with FD, a bZIP transcription factor, FT activates target genes, such as SUPPRESSOR OF OVEREXPRESSION OF CO (SOC1), FRUITFULL (FUL) and later APETALA1 (AP1), all of which encode MADS-box transcription factors. However, the floral transition involves a dramatic transcriptional reprogramming of the shoot meristem, and a complete picture of the global changes in gene expression occurring specifically in the SAM is still missing. Therefore, in the first part of this work, SAMs were specifically collected by the use of laser microdissection from plants experiencing a shift from SD to LD. RNA isolated from the meristems was converted to cDNA and gene expression quantified through next-generation sequencing by RNA-seq. Genes were grouped according to those increased or reduced in expression, with a particular focus on novel genes that were up-regulated similarly to SOC1 or FUL. Among them, the expression of a selected set of genes was tested by in situ hybridisation on wild-type apices to confirm their activation at the SAM, and to uncover their spatial pattern of mRNA expression. Several novel genes were confirmed to be induced by transferring plants to LD and they showed specific spatial patterns of expression in various regions of the SAM. Moreover, apices of ft tsf double mutants were also hybridised, to reveal whether those genes are induced by the photoperiodic cascade downstream of FT/TSF. Surprisingly, while many genes were induced only in the presence of FT/TSF, similarly to SOC1, some of them still respond to photoperiod in the ft tsf double mutants, suggesting that additional unknown signals may play a role in response to inductive day-length independently of FT and TSF. Further preliminary studies on a set of these novel genes are described in this study. In the second part, genetic approaches were employed to address the function of SHORT VEGETATIVE PHASE (SVP), which encodes a floral repressor of the MADS-box family, I demonstrating new interactions with floral promoter genes and distinct roles of the SVP gene in the leaves and in the meristem. II Zusammenfassung Der Vorgang bei welchem Angiospermen von vegetativem Wachstum zur Bildung von Blüten übergehen wird als Übergang zur Blüte („floral transition“) bezeichnet. Dieser entwicklungsbiologische Vorgang ist von großer Bedeutung und wird streng durch ein genetisches Netzwerk reguliert, wobei einige Komponenten des Netzwerkes auf Umweltfaktoren reagieren. Arabidopsis thaliana blüht früher unter Langtagbedingungen (LD) des Frühlings als unter den Kurztagbedingungen (SD) des Winters. Die Tageslänge oder Photoperiode ist einer der wichtigsten Umweltfaktoren welcher die Blühantwort beeinflusst. Die Photoperiode wird über die Blätter wahrgenommen während der Übergang zur Blüte im apikalen Sprossmeristem (SAM) stattfindet. Unter Langtagbedingungen wird eine genetische Kaskade im Leitgewebe des Blattes angestoßen, woraufhin ein Schlüsseltranskriptionsregulator mit dem Namen CONSTANS das Gen mit dem Namen FLOWERING LOCUS T (FT) und sein Homolog TWIN SISTER OF FT (TSF) aktiviert. Das FT Protein wird daraufhin durch das Phloem transportiert und erreicht schlussendlich das SAM wo es den Übergang zur Blüte auslöst. Durch Bildung eines Komplexes mit FD, einem bZIP Transkriptionsfaktor, aktiviert FT Zielgene wie SUPPRESSOR OF OVEREXPRESSION OF CO (SOC1), FRUITFULL (FUL) und später APETALA1 (AP1), welche für MADS-Box Transkriptionsfaktoren codieren. Der Übergang zur Blüte erfordert jedoch im SAM eine dramatische Neuprogrammierung der Transkriptionsvorgänge. Ein vollständiges Bild der Genexpression welche spezifisch im SAM stattfindet fehlt bislang. Daher wurden im ersten Teil dieser Arbeit apikale Sprossmeristeme durch Lasersezierung aus Pflanzen ausgeschnitten, welche von SD nach LD überführt worden waren. RNA, welche aus den Meristemen isoliert worden war, wurde in cDNA umgeschrieben und die Genexpression durch Next-Generation Sequencing durch RNA-seq quantifiziert. Die Gene wurden nach gesteigerter oder verringerter Expression sortiert, wobei ein besonderes Augenmerk auf neue Gene gelegt wurde deren Expression ähnlich der von SOC1 und FUL gesteigert wurde. Ein Teil dieser Gene wurde über in situ Hybridisierung in Wildtyp-Apizes getestet um ihre Aktivierung im SAM zu bestätigen und ihr räumliches Expressionsmuster aufzuklären. Für mehrere neue Gene konnte die Induktion durch Transfer der Pflanzen in LD bestätigt werden; auch zeigten sie spezifische räumliche Expressionsmuster in zahlreichen Regionen des apikalen Sprossmeristems. Es wurden auch Apizes von ft tsf Doppelmutanten hybridisiert um aufzudecken ob die neuen Gene an der von der Photoperiode abhängige Kaskade folgend auf FT/TSF beteiligt sind. Während viele Gene ähnlich wie SOC1 nur in Anwesenheit von FT/TSF induziert wurden, fanden sich erstaunlicherweise auch Gene welche auch in ft tsf Doppelmutanten noch auf die Photoperiode reagierten. Dies legt nahe, dass zusätzliche III unbekannte Signale eine Rolle in der Antwort auf eine induzierende Tageslänge unabhängig von FT und TSF spielen. In der vorliegenden Arbeit werden auch weitere Untersuchungen neuer Gene beschrieben. Im zweiten Teil der vorliegenden Arbeit wurden genetische Ansätze verwendet um die Funktion von SHORT VEGETATIVE PHASE (SVP), einem Blührepressor aus der MADS-Box Familie, zu untersuchen. Hierbei wurden neue Interaktionen mit die Blüte fördernden Genen und spezifische Rollen des SVP Gens in Blättern und Meristem aufgedeckt. IV Table of contents Abstract........................................................................................................................................ I Zusammenfassung....................................................................................................................... III Table of contents......................................................................................................................... V 1. Introduction 1.1 Plant development and the floral transition............................................................................ 1 1.2 Flowering pathways................................................................................................................ 2 1.2.1 Vernalisation pathway 1.2.2 Autonomous pathway 1.2.3 Gibberellin pathway 1.3 Photoperiodic pathway and activation of flowering............................................................... 7 1.3.1 Circadian clock 1.3.2 The photoperiodic cascade 1.3.2.1 GIGANTEA 1.3.2.2 CONSTANS 1.3.2.3 FT and TSF 1.4 The repression of flowering by FLC....................................................................................... 13 1.5 The repression of flowering by SVP....................................................................................... 14 1.6 The early floral transition: from vegetative meristem to inflorescence meristem.................. 17 1.6.1 The floral pathway integrators: classical and new members 1.6.1.1 FT as an integrator 1.6.1.2 FT and FD 1.6.1.3 SOC1 1.6.1.4 AGL24 1.6.1.5 LFY as an integrator 1.6.2 FUL in the floral transition 1.6.3 SPL genes: another chapter in the floral transition? 1.7 The later phase of the floral transition: from inflorescence meristem to floral meristem....... 27 1.7.1 Floral meristem identity genes 1.7.1.1 LFY 1.7.1.2 AP1 and CAL V 1.7.1.3 FUL 1.7.2 TFL1 1.7.3 Flower development and the ABC model 1.7.4 Flowering time genes regulate floral patterning 1.8 The shoot apical meristem: balance between stem cell maintainance and organ production. 33 1.9 Genomics approaches to study the floral transition................................................................ 35 1.9.1 “Single cell” technology 1.9.2 Next-generation-sequencing technologies and gene expression analysis 1.9.3 Recent genomics studies on SAM Objectives..................................................................................................................................... 41 2. Materials and methods........................................................................................................... 42 3. Global gene expression analysis of the floral transition at the SAM collected by LCM using next-generation sequencing........................................................................................................ 55 3.1 Defining the floral transition in Arabidopsis thaliana............................................................ 55 3.1.1 Shift and double shift experiments to study the floral transition 3.1.2 In situ hybridisation on marker genes 3.1.3 Double shift experiments to link expression of flowering time genes to floral commitment 3.2 Laser microdissection of shoot apical meristems................................................................... 65 3.2.1 Calculation of the material needed and choice of the techniques 3.2.2 Optimization of the protocol 3.2.3 Collection of the SAMs 3.2.4 RNA extraction from the captured material and RNA amplification 3.2.5 Single strand cDNA synthesis to test the RNA extracted from the meristems 3.2.6 Double strand cDNA synthesis for sequencing 3.3 Next-generation sequencing for gene expression analysis in the SAM.................................. 77 3.3.1 Deep sequencing with Illumina-Solexa and mapping of the short-sequence reads 3.3.2 Quality controls and reliability of the replicates 3.3.3 Use of housekeeping genes for the normalization 3.3.4 Expression values for known genes 3.3.5 Data analysis: identification of differentially expressed genes VI 3.3.6 GO term enrichment analysis 4. Characterization of novel genes induced during the floral transition selected from gene expression analysis.................................................................................................................... 93 4.1 Selection of the genes........................................................................................................... 93 4.2 Validation of the gene expression data: in situ hybridisations on candidate genes.............. 95 4.3 The use of mutants to test the response of the candidate genes to flowering pathways....... 101 4.4 D13: a lipid desaturase induced by photoperiod independently of FT and TSF.................. 103 4.4.1 On the expression pattern of D13: possible interactions with TFL1 4.4.2 On the signal inducing D13: triggering flowering without LD 4.4.3 Modulation of D13 expression with transgenic approaches 4.5 C11: a homeo-domain transcription factor which is dependent on FT/TSF......................... 111 4.5.1 C11 has close homologues which might play redundant roles with it 4.6 C19: a LRR protein responding to FT/TSF........................................................................... 113 4.6.1 Loss of function of C19 may influence flowering 4.7 Some bZIP transcription factors partially respond to FT/TSF............................................... 116 4.8 Several genes are related to the growth of the meristem in response to FT/TSF.................. 118 4.9 An unknown protein induced in the center of the SAM by FT/TSF..................................... 119 5. The role of SVP in leaf and meristem and the control of flowering time......................... 121 5.1 Loss of function of single floral promoter genes does not overcome the early flowering phenotype of svp mutants................................................................................... 121 5.2 SVP and the leaf: genetic and spatial interactions................................................................. 123 5.2.1 Role of SVP in the leaf and its relationship to FT and TSF 5.2.2 SOC1 in relation to SVP, FT and TSF. 5.2.3 Mis-expression of SVP in the leaf vasculature 5.3 SVP and the meristem: genetic and spatial interactions........................................................ 133 5.3.1 Role of SVP in the SAM and relation to SOC1 and FUL 5.3.2 Mis-expression of SVP in the meristem 5.3.3 Double shift experiments with mutants in flowering time genes 5.4 Dual role of SVP: leaf and meristem..................................................................................... 144 5.4.1 Quintuple mutant 5.4.2 Combined effect of mis-expression 5.5 Mis-expression of SVP.2 in different tissues......................................................................... 147 VII 6. Discussion................................................................................................................................ 149 6.1 Known genes and novel mechanisms that regulate the floral transition................................ 149 6.2 Next-generation sequencing for global gene expression analysis.......................................... 151 6.2.1 Comparison with available microarray data 6.2.2 General features of the data 6.3 Classification of the genes selected from the dataset............................................................. 154 6.4 Time of the response to the inducing photoperiod................................................................. 154 6.5 Spatial pattern of expression.................................................................................................. 155 6.6 Biological and molecular functions of the genes up-regulated during the floral transition... 157 6.6.1 Transcription factors and proteins involved in signaling 6.6.1.1 C19 6.6.1.2 C11 6.6.2 Additional layers of regulation 6.6.3 Genes involved in metabolism 6.6.4 Growth is a key process for the floral transition 6.6.4.1 Meristem growth 6.6.4.2 FT, “florigen”, and growth 6.7 Response to FT/TSF and responsiveness of ft tsf................................................................... 167 6.8 A novel signal identified in response to shift in photoperiod................................................. 168 6.9 SVP represses many genes...................................................................................................... 170 6.10 Is SVP a general repressor or is it downstream of photoperiodic induction?....................... 171 6.11 Double action leaf-meristem................................................................................................ 172 6.12 SVP and FLC........................................................................................................................ 172 6.13 A quintuple mutant reveals additional targets of SVP......................................................... 174 Conclusions................................................................................................................................. 175 Perspectives................................................................................................................................ 177 References.................................................................................................................................. 179 Appendix I.................................................................................................................................. 196 Appendix II................................................................................................................................ 201 Appendix III............................................................................................................................... 209 Acknowledgments...................................................................................................................... 211 Erklärung................................................................................................................................... 212 Lebenslauf.................................................................................................................................. 213 VIII

Description:
1.6 The early floral transition: from vegetative meristem to inflorescence Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-.
See more

The list of books you might like

Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.