The diploma thesis was aimed to explain the role of cytokinins in regulation of cotyledonary bud outgrowth in pea (Pisum sativum L.). Decapitation of the apex and subsequent application of IAA lanolin paste containing also BAP caused increased expression of genes PsDRM1 and PsBRC1 associated with dormancy. It suggests that cytokinins may have effect on the amount of transported auxin in the major stem. Application of cytokinin to the cotyledonary buds of intact plants activated their outgrowth. Imunolocalization showed polarization of PIN1 proteins in the treated buds, on contrary to untreated buds. It means that cytokinin directly influenced polarization of PIN1 proteins and the subsequent canalization of the polar auxin transport.

SummarySummarySummarySummary Cytokinins (CKs) are important group of plant hormones involved in a wide range of physiological and developmental processes. Endogenous levels of CKs as well as proportions of individual CK forms and derivatives are not constant and differ among plant species. The amounts of biological active CK forms (free bases and ribosides) are regulated through tangled machinery of metabolic conversions including biosynthesis, conjugation and degradation pathways. The main object of this thesis was to characterize the metabolic pathways involved in the regulation of bioactive CK levels in plants especially via CK biosynthesis with aspect to the environmental stimuli and via N- glucosylation pathway. It was shown, that light signal is an important input for modulating some CK-related genes and CK levels in Arabidopsis plants. The complex diurnal expression profiles of CK-biosynthetic genes (AtIPT1 - AtIPT9) in Arabidopsis plants indicated a strong dependence of AtIPT1 and AtIPT5 on light/dark phase in leaf rossetes. In contrast, no diurnal oscillation of AtIPT transcript levels was recorded in roots. Although the content of endogenous CKs was not constant in plants and varied during a day, no statistically significant correlation between light/dark cycle and oscillation in CK levels was...

Am Beispiel von Aster alpinus 'Happy End' wird der Entwicklungsverlauf vom Zeitpunkt der Vermehrung bis zur Ausbildung von Infloreszenzen beschrieben, um daraus Empfehlungen für die Kultur als blühende Topfstaude abzuleiten. Dazu wurden über einen Zeitraum von drei Jahren Versuche mit unterschiedlich vermehrten Pflanzen durchgeführt. Verwendet wurden hierbei Pflanzen aus generativer und vegetativer Vermehrung (Stecklinge, in vitro). Untersucht wurde der Verlauf der vegetativen und generativen Entwicklung unter spezieller Berücksichtigung der Dormanz und der Differenzierung der Blütenanlagen. Die Umstimmung des vegetativen Meristems in ein generatives wurde durch die Transplantation von Sproßknospen in die Invitro-Kultur und durch histologische Untersuchungen erfaßt. Während der juvenilen Entwicklungsphase und vor dem Eintritt in die Endodormanz erfolgt die Blüteninduktion. Der früheste Zeitpunkt der Blütendifferenzierung konnte bei Aster alpinus Ende September festgestellt werden. Die Endodormanz beginnt ab Mitte Oktober und dauert bis Mitte-Ende Dezember an. Erst danach ist eine weitere Entwicklung der Infloreszenz möglich. Anschließend wurde der Einfluß der exogenen Applikation mit dem Cytokinin 6-Benzylaminopurin und der Einfluß der Kühlung auf die vegetative und generative Entwicklung untersucht. Dazu wurden die Pflanzen während der juvenilen Phase über einen Zeitraum von 6 Wochen mit BAP-Lösungen in unterschiedlichen Konzentrationen (15, 20 und 25 mg/l) besprüht. Der Einfluß der Kühlung wurde durch die Erfassung von Freiland temperaturen und durch eine zeitweise Aufstellung der Pflanzen im Kühlraum ermittelt. Erfaßt wurden dabei alle Temperaturen unter 2ø C und die Dauer der Kühlung. Die Applikation von BAP in einer Konzentration von 20 und 25 mg/l bewirkte eine signifikant höhere Anzahl gestreckter, vegetativer Sproßknospen. Der Einfluß der Cytokininbehandlung auf die Anzahl der gestreckten Infloreszenzen war dagegen nicht signifikant. Die Wirkung der Kühlung auf die Streckung von Infloreszenzen erwies sich gegenüber der Cytokininbehandlung als wesentlich stärker. Die Dauer der Kühlung korreliert positiv mit der Anzahl gestreckter Infloreszenzen. Das Vernalisationsbedürfnis ist jedoch nicht obligat. Beide Faktoren (Cytokininapplikation und Kühlung) zusammen, haben eine synergistische Wirkung auf die Anzahl gestreckter Infloreszenzen. / A study was made on the development rhythm of perennials on Aster alpinus 'Happy End' from the beginning of propagation until flowering, to give recommondation for cultivation as a flowering potplant. Approximately three years of examinations with different propagated plants were carried out. Plants from generative and vegetative propagation (cuttings, in vitro) were used. The course of vegetative and generative development was examined especially taking into account the dormant stages and the differentation of flowering plants. The change of the vegetative to the generative meristem was recorded by the transplantation of shoots to the invitro-culture and by histological examinations. The flower initiation took place during the juvenile development phase and before the beginning of endodormancy. The earliest flower differentation was observed at the end of September. The endodormancy started in the middle of october and lasted until mid to late December. Only afterwards was a further development of the inflorescence possible. In the second part the influence of exogen application with the cytokinin 6-Benzylaminopurin (BAP) and the influence of chilling at the vegetative and generative development were examined. In addition the plants were sprayed with BAP-solutions with different concentrations during the juvenile phase of about 6 weeks. The influence of chilling was measured under outdoor conditions and with temporary artificial chilling. All temperatures under 2 øC and the length of time they were chilled, were recorded. Afterwards the plants were maintained in the greenhouse until flowering. The application of BAP with concentrations of 20 and 25 mg/l caused significantly longer vegetative shoots. The influence of the cytokinin treatment on the number of elongated inflorescences was non-significant in contrast. The rate of progress to generative development improved with increased chilling time. The vernalisation response is not obligatory. Both factors (cytokinin application and chilling) together have a synergistic effect on the number of elongated inflorescences.

Licht und Pflanzenhormone sind essentiell für das Wachstum und die Entwicklung von Pflanzen. Es ist nur wenig darüber bekannt, wie sie die Transkription organellärer Gene beeinflussen. In Arabidopsis thaliana gibt es drei kernkodierte Phagentyp-RNA-Polymerasen (RpoT), welche für die organelläre Transkription verantwortlich sind. Diese werden in die Plastiden (RpoTp), die Mitochondrien (RpoTm) oder zu beiden Organellen (RpoTmp) transportiert. Neben den beiden kernkodierten RNA-Polymerasen (NEP) existiert in den Plastiden eine plastidärkodierte RNA-Polymerase (PEP), welche zusätzliche Sigmafaktoren zur Promotererkennung benötigt. Um die Lichtabhängigkeit der Expression der RpoT Gene sowie NEP-transkribierter Chloroplastengene zu analysieren, wurde die Akkumulation von RpoT- und rpoB-Transkripten in 7-Tage alten Keimlingen unter verschiedenen Lichtbedingungen mittels quantitativer real-time PCR untersucht. Die Änderungen in der Transkriptakkumulation deuten darauf hin, dass rote, blaue und grüne Wellenlängen die Expression der drei RpoT Gene unterschiedlich stark stimulieren. Untersuchungen an verschiedenen Lichtrezeptor-Mutanten zeigten, dass die Lichtinduktion der RpoT Genexpression überaus komplex ist und ein interagierendes Netzwerk aus multiplen Rezeptoren und Transkriptionsfaktoren an der Signalweiterleitung beteiligt ist. Das Phytohormon Cytokinin wird durch Histidin Kinase Rezeptoren (AHK) detektiert. Es gibt drei unterschiedliche Rezeptoren: AHK2, AHK3 und AHK4. Diese sind Teil eines Zwei-Komponenten-Systems, welches Signale mit Hilfe einer Phosphorylierungskette überträgt. Der Einfluss von Cytokinin auf die plastidäre Transkription wurde mit Hilfe von Cytokininrezeptor-Mutanten untersucht, um die Funktion von AHK2, AHK3 und AHK4 zu analysieren. Um weitere Informationen darüber zu erhalten, wie die plastidäre Transkription durch PEP mittels Cytokinin reguliert wird, wurden die Hormoneffekte auf die plastidäre Transkription in Sigmafaktor-Mutanten untersucht. / Light and plant hormones are essential for plant growth and development. Only little information is available about how these signals influence the transcription of organellar genes. Arabidopsis thaliana possesses three nuclear-encoded phage-type RNA polymerases (RpoT) for organellar transcription. They are imported into plastids (RpoTp), mitochondria (RpoTm), or into both organelles (RpoTmp). Besides the two nuclear-encoded plastid polymerases (NEP), plastids contain an additional plastid-encoded RNA polymerase (PEP), which needs additional sigma factors for promoter recognition. Interested in the expression of RpoT genes and NEP-transcribed plastid genes in response to light we analyzed transcript levels of RpoT and rpoB genes in 7-day-old wild-type plants under different light conditions by quantitative real-time-PCR. The observed changes in transcript accumulation indicated that red, blue, and green light differentially stimulated the expression of all three RpoT genes. Further analyses using different photoreceptor mutants showed that light induction of RpoT gene expression is surprisingly complex based on a network of multiple photoreceptors an d downstream pathways. Cytokinin signals are perceived by the histidine kinase (AHK) receptor family. There exist three different membrane-bound receptors: AHK2, AHK3 and AHK4/CRE1. These receptors are part of a two-component signaling system which transfers signals via phosphorelay mechanisms. Interested in the potential role of AHK2, AHK3 and AHK4/CRE1 in the transduction of cytokinin signals into the chloroplast, we analyzed the influence of cytokinin on plastidial transcription in receptor mutants. To gain more information on how plastid transcription by PEP is regulated by cytokinin, the influence of cytokinin in sigma factor mutants was also studied.

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The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.

No description available.

No description available.

Mitogen-activated protein kinases are part of an evolutionarily conserved protein phosphorylation cascade which serves essential regulatory functions in eukaryotic organisms. Although the role of MAPKs in the regulation of a plant’s response to environmental stress and plant defense has been well established, very little is known about their role in the regulation of plant developmental processes. In order to examine the role of MAPKs in plant growth and development, a strong mammalian MAPK phosphatase (MKP-1), which is known to inactivate MAPKs in plants, was introduced into tobacco plants. In tobacco plants, MKP-1 overexpression altered plant responses to the phytohormones, ethylene and cytokinin. Tobacco plants expressing MKP-1 flowered earlier and senesced later than wild-type. Additionally, these plants exhibited similar floral morphology as flowers from ethylene-insensitive tobacco plants. These observed phenotypes seem to depend on the protein phosphatase activity, as transgenic lines expressing an inactive form of MKP-1 (MKPCS) did not show the same phenotypes. Furthermore, both tobacco and Arabidopsis MKP-1 transgenic plants exhibited increased shoot regeneration when compared to wild-type plants, suggesting increased cytokinin sensitivity. In an attempt to elucidate the mechanism by which MKP-1 affects plant growth and development, expression of selected genes were analyzed using RT-PCR. MKP-1 transformed tobacco plants exhibited downregulated expression of an ethylene biosynthesis gene (NtACO) and upregulated expression of a pathogenesis-related gene (PR-1b), similar to gene expression studies previously conducted in plants with increased production of cytokinin. The same MKP-1 transgenic plants also exhibited upregulated expression of the flowering time gene, FT. Results from this study indicate that constitutive expression of MKP-1 may interfere with ethylene-related MAPK pathways, which normally serves to restrict plant growth during times of environmental stress. The reduced responses to ethylene resulted in elevated sensitivity to cytokinin, promoting an enhanced shoot regeneration phenotype.

Os hormônios auxina e citocininas são essenciais ao desenvolvimento das plantas, pois controlam os processos de divisão, expansão e diferenciação celular, os quais, por sua vez, influenciam desde a formação do embrião até o amadurecimento dos frutos e senescência. Auxinas e citocininas regulam respostas fisiológicas comuns, sugerindo haver múltiplos mecanismos de interação. Neste trabalho, um modelo para estudar a interação entre auxinas e citocininas no controle do desenvolvimento é proposto, sendo baseado em plantas mutantes e transgênicas, além de duplos mutantes, com alterações na sensibilidade ou metabolismo de auxina e citocinina. O mutante bushy root (brt) foi caracterizado como pouco sensível à citocinina e sugere uma importante função da citocinina no desenvolvimento da semente e na determinação da dominância apical em tomateiro. Os mutantes potato leaf (c) e entire (e) foram introgredidos no background Micro-Tom (MT), caracterizados e utilizados para verificar como eles afetam a sensibilidade à auxina e como interferem no desenvolvimento da planta. Esses mutantes foram comparados com MT, com o mutante diageotropica (dgt), que é pouco sensível à auxina, e com os duplos mutantes c dgt e dgt e. A mutação c não alterou a sensibilidade à auxina e estaria influenciando apenas a arquitetura foliar. O mutante e parece ser mais sensível à auxina com relação à capacidade de formar raízes in vitro e partenocarpia. O duplo mutante dgt e mostrou fenótipo aditivo (intermediário), sugerindo que DGT e E agem em vias paralelas na resposta à auxina. Foram criadas linhagens transgênicas com superexpressão da enzima citocinina oxidase de Arabidopsis (35S:AtCKX2), que resulta em plantas com baixos níveis endógenos de citocinina. As linhagens transgênicas (MT CKX2 e dgt CKX2) foram comparadas com MT, brt, dgt e brt dgt. A maioria dos parâmetros que estavam relacionados com o desenvolvimento vegetativo, e todos os parâmetros relacionados ao desenvolvimento reprodutivo, foram associados ao efeito dos níveis absolutos de auxina e citocinina, sendo provavelmente resultantes de alterações nos processos de divisão e expansão celular. A dominância apical e a morfogênese in vitro (formação de gemas caulinares e raízes) foram associadas ao efeito do balanço auxina/citocinina, sendo esses processos conhecidamente regulados pelo efeito dessas classes hormonais na diferenciação celular / The plant hormones auxin and cytokinin are crucial for plant development, since they control cell division, expansion and differentiation, which regulate developmental processes starting from embryo formation to fruit ripening and senescence. Auxins and cytokinins usually regulate the same physiological responses, suggesting multiple mechanisms of interaction between these hormones. In this work, a model to study auxin and cytokinin interaction in the control of plant development is proposed, based on mutants, transgenic lines and double mutants with alterations on the auxin and cytokinin sensitivity or metabolism. The bushy root (brt) mutant was characterized as having low cytokinin sensitivity and suggested a relevant function to cytokinin in the seed development and apical dominance in tomato. The potato leaf (c) and entire (e) mutants were introgressed in the Micro-Tom background, characterized and used to check how they affect the auxin sensitivity and the plant development. The responses from these mutants were compared to MT and diageotropica (dgt), which is a low sensitive auxin mutant, and also compared to c dgt and dgt e double mutants. The c mutation did not alter the auxin sensitivity and was only related to leaf architecture. The e mutant seemed to be more sensitive to auxin in the in vitro root induction and parthenocarpy. The double mutant dgt e showed an additive phenotype, suggesting that DGT and E act in parallel pathways controlling auxin sensitivity. Transgenic lines were generated overexpressing the cytokinin oxidase from Arabidopsis (35S:AtCKX2), which renders plants with low endogenous cytokinin levels. Transgenic lines (MT CKX2 and dgt CKX2) were compared to MT, brt, dgt and brt dgt. Most of the traits related to vegetative development and all traits related to reproductive development were linked to the effect of auxin and cytokinin absolute levels, probably reflecting to the effect of these hormones in cell division and expansion. Apical dominance and in vitro morphogenesis (shoot and root formation) were linked to the auxin-to-cytokinin ratio, since these processes are well known to be regulated by the effect of these two hormones in cell differentiation