TY - THES A1 - Guedes Corrêa, Luiz Gustavo T1 - Evolutionary and functional analysis of transcription factors controlling leaf development T1 - Evolutionäre und funktionelle Analyse von Transkriptionsfaktoren, welche die Blattentwicklung steuern N2 - Leaves are the main photosynthetic organs of vascular plants, and leaf development is dependent on a proper control of gene expression. Transcription factors (TFs) are global regulators of gene expression that play essential roles in almost all biological processes among eukaryotes. This PhD project focused on the characterization of the sink-to-source transition of Arabidopsis leaves and on the analysis of TFs that play a role in early leaf development. The sink-to-source transition occurs when the young emerging leaves (net carbon importers) acquire a positive photosynthetic balance and start exporting photoassimilates. We have established molecular and physiological markers (i.e., CAB1 and CAB2 expression levels, AtSUC2 and AtCHoR expression patterns, chlorophyll and starch levels, and photosynthetic electron transport rates) to identify the starting point of the transition, especially because the sink-to-source is not accompanied by a visual phenotype in contrast to other developmental transitions, such as the mature-to-senescent transition of leaves. The sink-to-source transition can be divided into two different processes: one light dependent, related to photosynthesis and light responses; and one light independent or impaired, related to the changes in the vascular tissue that occur when leaves change from an import to an export mode. Furthermore, starch, but not sucrose, has been identified as one of the potential signalling molecules for this transition. The expression level of 1880 TFs during early leaf development was assessed by qRTPCR, and 153 TFs were found to exhibit differential expression levels of at least 5-fold. GRF, MYB and SRS are TF families, which are overrepresented among the differentially expressed TFs. Additionally, processes like cell identity acquisition, formation of the epidermis and leaf development are overrepresented among the differentially expressed TFs, which helps to validate the results obtained. Two of these TFs were further characterized. bZIP21 is a gene up-regulated during the sink-to-source and mature-to-senescent transitions. Its expression pattern in leaves overlaps with the one observed for AtCHoR, therefore it constitutes a good marker for the sink-to-source transition. Homozygous null mutants of bZIP21 could not be obtained, indicating that the total absence of bZIP21 function may be lethal to the plant. Phylogenetic analyses indicate that bZIP21 is an orthologue of Liguleless2 from maize. In these analyses, we identified that the whole set of bZIPs in plants originated from four founder genes, and that all bZIPs from angiosperms can be classified into 13 groups of homologues and 34 Possible Groups of Orthologues (PoGOs). bHLH64 is a gene highly expressed in early sink leaves, its expression is downregulated during the mature-to-senescent transition. Null mutants of bHLH64 are characterized by delayed bolting when compared to the wild-type; this indicates a possible delay in the sink-to-source transition or the retention of a juvenile identity. A third TF, Dof4, was also characterized. Dof4 is neither differentially expressed during the sink-to-source nor during the senescent-to-mature transition, but a null mutant of Dof4 develops bigger leaves than the wild-type and forms a greater number of siliques. The Dof4 null mutant has proven to be a good background for biomass accumulation analysis. Though not overrepresented during the sink-to-source transition, NAC transcription factors seem to contribute significantly to the mature-to-senescent transition. Twenty two NACs from Arabidopsis and 44 from rice are differentially expressed during late stages of leaf development. Phylogenetic analyses revealed that most of these NACs cluster into three big groups of homologues, indicating functional conservation between eudicots and monocots. To prove functional conservation of orthologues, the expression of ten NAC genes of barley was analysed. Eight of the ten NAC genes were found to be differentially expressed during senescence. The use of evolutionary approaches combined with functional studies is thus expected to support the transfer of current knowledge of gene control gained in model species to crops. N2 - Das Blatt ist das wichtigste photosynthetische Organ von Gefäßpflanzen und die Blattentwicklung ist von einer exakten Genexpression abhängig. Transkriptionsfaktoren (TFs) sind globale Regulatoren der Genexpression. Diese sind, in fast allen biologischen Vorgängen der Eukaryoten, von grundlegender Bedeutung. Das Promotionsarbeit legte den Schwerpunkt auf den sogenannten Sink-source-Übergang in Blättern der Modellpflanze Arabidopsis thaliana, zu deutsch Ackerschmalwand. Ein besonderer Fokus lag dabei auf der Analyse von TFs, welche eine wichtige Rolle in der frühen Blattentwicklung spielen. Sehr junge Blätter befinden sich im sogenannten Sink-Status, sie müssen Photoassimilate aus älteren, sogenannten Source-Blättern importieren, da sie selbst noch nicht in der Lage sind, hinreichend viel Kohlendioxid über die Photosynthese zu binden. Der Übergang vom Sink- in den Source-Zustand eines Blattes ist ein hoch komplizierter biologischer Prozess, der bisher nur in Ansätzen verstanden ist. Im Rahmen der Doktorarbeit wurden molekulare und physiologische Marker identifiziert, die es erlauben, den für das bloße Auge nicht ohne weiteres sichtbaren Sink-Source-Übergang zu erkennen. Dazu wurde beispielsweise die Aktivität bestimmter Gene, unter anderem der Gene AtSUC2 und AtCHoR, mittels molekularer Techniken verfolgt. Um den Über zwischen den beiden Entwicklungszuständen eingehend zu charakterisieren wurde die Aktivität von etwa 1900 Regulatorgenen mittels eines multiparallelen Verfahrens - der sogenannten quantitativen RT-PCR - untersucht. Bei den Regulatoren handelt es sich um Transkriptionsfaktoren, die die Aktivität anderer Gene der Pflanzen steuern. Von allen untersuchten Genen zeigten 153 ein vom Blattstadium abhängiges Aktivitätsmuster. Dabei waren Mitglieder der GRF, MYB und SRS Familien überrepräsentiert. Für die gefundenen Transkriptionsfaktoren zeigte sich besonders häufig eine Assoziation zu Prozessen wie Spezialisierung von Zellen, Entwicklung der Epidermis sowie der Blattentwicklung. Zwei ausgewählte Regulatorproteine - bZIP21 und bHLH64 - wurden detaillierter charakterisiert. Das bZIP21-Gen zeigte eine starke Aktivität whrend des Sink-Source-Übergangs. Sein Expressionsmuster in Blättern deckt sich mit dem für AtCHoR beobachteten Expressionsmuster, so dass bZIP21 als ein neuer Marker für die Sink-Source- Transition dienen kann. Es konnten keine homozygoten Null-Mutanten des Gens erhalten werden, was die Vermutung nahelegt, dass gänzliche Abwesenheit von bZIP21 letal fr die Pflanze sein kann. Phylogenetische Analysen ergaben, dass bZIP21 ortholog zum Gen Liguleless2 aus Mais ist. In diesen Analysen konnte gezeigt werden, dass alle pflanzlichen bZIP Transkriptionsfaktoren von vier Gründergenen abstammen und alle bZIPs der Angiospermen in 13 homologe Klassen und 34 mögliche orthologe Klassen (Possible Groups of Orthologues, PoGOs) eingeordnet werden können. Das bHLH64 Gen ist im unreifen Blatt stark aktiv und während des Alterungsprozesses herunterreguliert. Null-Mutationen von bHLH64 zeigen eine verzögerte Blütenbildung im Vergleich zum Wildtyp; dies weist auf eine mögliche Verzögerung in des Sink-SourceÜbergangs oder Aufrechterhaltung der jugendlichen Identität hin. Ein dritter Transkriptionsfaktor, Dof4, wurde ebenfalls charakterisiert. Dof4 wird weder während des Sink-Source-Übergangs noch während des Alterungsprozesses unterschiedlich exprimiert. Eine Null-Mutante von Dof4 besaß größere Blätter und eine höhere Anzahl an Schoten in Vergleich zum Wildtyp. Diese Mutanten erwiesen sich als gut geeignet fr die Analyse der Akkumulation pflanzlicher Biomasse. Obwohl während der Sink-Source Transition nicht überrepräsentiert, scheinen NAC Transkriptionsfaktoren eine große Rolle während des Alterungsprozesses zu spielen. Zweiundzwanzig NAC-Gene von Arabidopsis und 44 von Reis sind in der späten Phase der Blattentwicklung verändert exprimiert. Phylogenetische Analysen erlaubten die Einordnung der meisten dieser NACs in vier homologe Gruppen, was auf einen funktionellen Erhalt zwischen einkeimblättrigen und zweikeimblättrigen Pflanzen hinweist. Um den funktionellen Erhalt von Orthologen zu untersuchen, wurde die Expression von zehn NAC-Genen aus Gerste analysiert. Acht dieser Gene zeigten eine von der Blattalterung abhängige Expression. Die Kombination von evolutionären Analysen und funktionellen Studien könnte den Wissenstransfer von Modellpflanzen auf Getreidepflanzen in Zukunft vereinfachen. KW - Evolution KW - Transkriptionsfaktoren KW - Pflanzen KW - Entwicklung KW - Blatt KW - evolution KW - transcription factors KW - plant KW - development KW - leaf Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-40038 ER - TY - JOUR A1 - Arvidsson, Samuel Janne A1 - Perez-Rodriguez, Paulino A1 - Müller-Röber, Bernd T1 - A growth phenotyping pipeline for Arabidopsis thaliana integrating image analysis and rosette area modeling for robust quantification of genotype effects JF - New phytologist : international journal of plant science N2 - To gain a deeper understanding of the mechanisms behind biomass accumulation, it is important to study plant growth behavior. Manually phenotyping large sets of plants requires important human resources and expertise and is typically not feasible for detection of weak growth phenotypes. Here, we established an automated growth phenotyping pipeline for Arabidopsis thaliana to aid researchers in comparing growth behaviors of different genotypes. The analysis pipeline includes automated image analysis of two-dimensional digital plant images and evaluation of manually annotated information of growth stages. It employs linear mixed-effects models to quantify genotype effects on total rosette area and relative leaf growth rate (RLGR) and ANOVAs to quantify effects on developmental times. Using the system, a single researcher can phenotype up to 7000 plants d(-1). Technical variance is very low (typically < 2%). We show quantitative results for the growth-impaired starch-excessmutant sex4-3 and the growth-enhancedmutant grf9. We show that recordings of environmental and developmental variables reduce noise levels in the phenotyping datasets significantly and that careful examination of predictor variables (such as d after sowing or germination) is crucial to avoid exaggerations of recorded phenotypes and thus biased conclusions. KW - development KW - growth KW - leaf area KW - modeling KW - phenotyping Y1 - 2011 U6 - https://doi.org/10.1111/j.1469-8137.2011.03756.x SN - 0028-646X VL - 191 IS - 3 SP - 895 EP - 907 PB - Wiley-Blackwell CY - Malden ER - TY - JOUR A1 - Schmidt, Romy A1 - Schippers, Jos H. M. A1 - Mieulet, Delphine A1 - Obata, Toshihiro A1 - Fernie, Alisdair R. A1 - Guiderdoni, Emmanuel A1 - Müller-Röber, Bernd T1 - Multipass, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways JF - The plant journal N2 - Growth regulation is an important aspect of plant adaptation during environmental perturbations. Here, the role of MULTIPASS (OsMPS), an R2R3-type MYB transcription factor of rice, was explored. OsMPS is induced by salt stress and expressed in vegetative and reproductive tissues. Over-expression of OsMPS reduces growth under non-stress conditions, while knockdown plants display increased biomass. OsMPS expression is induced by abscisic acid and cytokinin, but is repressed by auxin, gibberellin and brassinolide. Growth retardation caused by OsMPS over-expression is partially restored by auxin application. Expression profiling revealed that OsMPS negatively regulates the expression of EXPANSIN (EXP) and cell-wall biosynthesis as well as phytohormone signaling genes. Furthermore, the expression of OsMPS-dependent genes is regulated by auxin, cytokinin and abscisic acid. Moreover, we show that OsMPS is a direct upstream regulator of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3, OsEXPB6 and the endoglucanase genes OsGLU5 and OsGLU14. The multiple responses of OsMPS and its target genes to various hormones suggest an integrative function of OsMPS in the cross-talk between phytohormones and the environment to regulate adaptive growth. KW - development KW - expansin KW - transcription KW - Oryza sativa KW - hormone KW - abiotic stress Y1 - 2013 U6 - https://doi.org/10.1111/tpj.12286 SN - 0960-7412 SN - 1365-313X VL - 76 IS - 2 SP - 258 EP - 273 PB - Wiley-Blackwell CY - Hoboken ER - TY - GEN A1 - Huynen, Leon A1 - Suzuki, Takayuki A1 - Ogura, Toshihiko A1 - Watanabe, Yusuke A1 - Millar, Craig D. A1 - Hofreiter, Michael A1 - Smith, Craig A1 - Mirmoeini, Sara A1 - Lambert, David M. T1 - Reconstruction and in vivo analysis of the extinct tbx5 gene from ancient wingless moa (Aves: Dinornithiformes) T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background The forelimb-specific gene tbx5 is highly conserved and essential for the development of forelimbs in zebrafish, mice, and humans. Amongst birds, a single order, Dinornithiformes, comprising the extinct wingless moa of New Zealand, are unique in having no skeletal evidence of forelimb-like structures. Results To determine the sequence of tbx5 in moa, we used a range of PCR-based techniques on ancient DNA to retrieve all nine tbx5 exons and splice sites from the giant moa, Dinornis. Moa Tbx5 is identical to chicken Tbx5 in being able to activate the downstream promotors of fgf10 and ANF. In addition we show that missexpression of moa tbx5 in the hindlimb of chicken embryos results in the formation of forelimb features, suggesting that Tbx5 was fully functional in wingless moa. An alternatively spliced exon 1 for tbx5 that is expressed specifically in the forelimb region was shown to be almost identical between moa and ostrich, suggesting that, as well as being fully functional, tbx5 is likely to have been expressed normally in moa since divergence from their flighted ancestors, approximately 60 mya. Conclusions The results suggests that, as in mice, moa tbx5 is necessary for the induction of forelimbs, but is not sufficient for their outgrowth. Moa Tbx5 may have played an important role in the development of moa’s remnant forelimb girdle, and may be required for the formation of this structure. Our results further show that genetic changes affecting genes other than tbx5 must be responsible for the complete loss of forelimbs in moa. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1117 KW - tbx5 KW - Moa KW - gene expression KW - ancient DNA KW - development KW - forelimb Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431599 SN - 1866-8372 IS - 1117 ER -