Dokument: Funktionale Charakterisierung und Analyse des Promotors von ARABIDOPSIS CRINKLY 4

Titel:	Funktionale Charakterisierung und Analyse des Promotors von ARABIDOPSIS CRINKLY 4
Weiterer Titel:	Functional characterization and analysis of the ARABIDOPSIS CRINKLY 4 promoter
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=38814
URN (NBN):	urn:nbn:de:hbz:061-20160707-095239-2
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Hülsewede, Adrian [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 6,87 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 27.06.2016 / geändert 27.06.2016
Beitragende:	Prof. Dr. Simon, Rüdiger [Gutachter] Prof. Dr. Feldbrügge, Michael [Gutachter]
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	In this thesis, the 5’ regulatory sequence of ACR4 was analyzed with molecular and computational methods to identify specific sequence motifs that control gene expression in the Arabidopsis root. It is shown that the promoter of ACR4 has a modular organization, comprising 4 modules, whereby each module is needed for a component of the total expression pattern. Module 1 is needed for epidermal expression and module 2 is needed for expression in the distal root meristem (DRM). Module 3 is enhancing ACR4 expression in CSCs and module 4 is a general enhancer of protein expression. Through a process of elimination, the location of module 1 could be mapped to a 241 bp long region (elements E9 and E10) and module 4 consists of the 483 bp long 5’ UTR. This UTR contains four upstream open reading frames, which are likely inhibiting protein translation. While the exact location of module 3 could not be determined, module 2 could be narrowed down to a combination of two evolutionary conserved motifs, each 15 bp in length and spaced by 66bp. These two motifs are acting redundantly and are necessary for robust expression of ACR4 in the DRM. The 150 bp long element E7, containing these motifs, is sufficient for expression of ACR4 in the DRM, when combined with a core 35S CaMV promoter. Since ACR4 expression is upregulated in the RAM after synthetic CLE40-peptide treatment, it was investigated if the ACR4 RLK is involved in an auto-regulatory feedback loop. It could be shown that the RLK is needed for this process, but not necessarily its kinase domain. Thus, factors, which are directly regulating ACR4 gene expression, can be both, upstream and downstream of ACR4 signaling. This potential auto-regulatory function was then examined in closer detail and also with regard to ACR4 expression strength in mutants of other known players in the CLE40-ACR4-WOX5 signaling module, namely wox5-1, acr4-2 and cle40-2. Here it was found, that although ectopic overexpression of WOX5 and global treatment with synthetic CLE40 peptide both lead to an upregulation of ACR4 expression in the RAM, neither WOX5 nor CLE40 or ACR4 are required for basal expression of ACR4 in the DRM. Furthermore, in this thesis, transcriptions factors were identified which were able to directly regulate ACR4 expression, and a first analysis of the expression patterns of the ACR4-homologs CRR1 and CRR3 was performed to identify a potential overlap of the expression domain, which could hint to a functional redundancy between these CRINKLY family members. Here, the previous observation that WOX5 is not a direct regulator of ACR4 transcription is supported by the finding that WOX5 was not found as a protein binding to the ACR4 promoter in a protoplast transactivation system (PTA). Additionally, WOX5 was not influencing luciferase expression in Nicotiana benthamiana epidermal cells and a promoter version with a mutated WUS binding site gave rise to a wild type ACR4 expression pattern. To find transcription factors, which are regulating ACR4 expression, a PTA and a yeast-one-hybrid (Y1H) screen were combined. In these experiments several transcription factors were identified which were able to influence reporter expression. From those proteins, the ability to influence ACR4 expression could be confirmed for a transcription factor, which was named BINDING TO THE PROMOTER OF ACR4 (BTA). In a luciferase assay in N. benthamiana BTA, a potential zinc-finger protein, was able to downregulate ACR4 expression. The expression patterns of ACR4 and BTA are partially overlapping in the root cap, which was shown with a promoter-reporter construct. Thus it seems likely that BTA may regulate ACR4 expression in its native expression domain. In contrast to BTA, the expression domains of CRR1 and CRR3 are complementary to the expression domain ACR4. While CRR1 is expressed in the epidermis and the cortex, the expression of CRR3 is restricted to the LRC. Thus it seems unlikely that both genes are acting redundantly together with ACR4 in regulating CSC homoeostasis. ACR4 orthologs exists in all land plant species examined but not in green algae. This suggest that the ACR4 gene is an evolutionary innovation, which evolved after the transition step from aquatic living plants to land plants.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie
Dokument erstellt am:	07.07.2016
Dateien geändert am:	07.07.2016
Promotionsantrag am:	18.01.2016
Datum der Promotion:	18.03.2016