During embryogenesis, the architecture of the plant and the food reserves for seed germination are established. Abscisic acid (ABA) regulates seed development and dormancy. It controls genes involved in stress responses. ABA-responsive basic leucine zipper (bZIP) transcription factors are identified by interaction with ABA responsive cis-regulatory elements. The transcription factor ABI5 is one of these. It regulates gene expression during embryogenesis and in response to ABA. An ABA-insensitive mutant, abi5-6, exhibits no gross morphological defects other than the effect on seed germination in the presence of ABA. Thus, microarray analysis was employed to search for molecular phenotypes. We used cDNA microarrays to analyze ABA regulated gene expression and the role of ABI5 in seedlings. 310 genes were identified as ABI5/ABA regulated genes. 161 of these genes were regulated by ABI5, and 134 of ABI5-regulated genes were co-regulated by ABA. Only a small number of genes altered expression in both Pro35S:ABI5 and abi5-6 genetic backgrounds indicating the preferential binding of the bZIP protein dimers to specific promoter sequences. To determine the optimal platform for identifying ABI5-regulated genes in seeds, a cDNA microarray, the Agilent Arabidopsis Oligo microarray, and the Affymetrix ATH1 arrays were tested. Cross platform comparisons utilized 4,518 genes present on all three platforms. The best correlation was between the Agilent and the Affymetrix results. Furthermore, the Affymetrix results correlated best with qRT-PCR validation data for selected genes. A small number of genes including AtCOR413 pm-1 showed a consistent expression pattern across the three platforms. A robust ABRE cis-regulatory element was identified in the promoter of AtCOR413 pm-1. Further studies showed binding of ABI5 to the promoter of AtCOR413 pm-1 by Electrophoretic Mobility Shift Assays (EMSA) and validated the expression of ABI5 and AtCOR413 pm-1 in abi5-6 seeds by qRT-PCR and RNA gel blot analysis. Transactivation assays using AtCOR413 pm-1 promoter:GUS fusions in Arabidopsis dry seed and seedlings revealed ABI5 acts as a negative regulator for AtCOR413 pm-1 in dry seeds, while other proteins may play major roles in regulating responses to ABA and low temperature (LT) in seedlings.

Orientador: Michel Georges Albert Vincentz / Tese (outorado) - Universidade Estaulal de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T10:23:29Z (GMT). No. of bitstreams: 1 Tomaz_JuarezPires.pdf: 3125737 bytes, checksum: 8b543a90ff7edc4df818dc09a5ea0bc5 (MD5) Previous issue date: 2008 / Resumo: Na planta modelo eudicotiledónea A. thaliana quatro genes para fatores de transcrição do tipo bZIP que são homólogos a Opaco-2 (O2) do milho, uma monocotiledônea, foram identificados. O2 é um regulador chave do metabolismo coordenado de carbono e nitrogênio e da síntese de prolaminas de reserva durante o desenvolvimento da semente. Estes quatro genes, AtbZIP9, o par de parálogos AtbZIP10 e AtbZIP25, e AtbZIP63, o provável ortólogo de O2, formam o Grupo C de genes bZIP de Arabidopsis. Sabe-se que AtbZIP9 provavelmente desempenhe um papel no processo de desenvolvimento do floema, AtbZIP10 está associado com e resposta à estresses, além de, junto com AtbZIP25, participar na regulação de genes de proteínas de reserva na semente e que AtbZIP63 pode estar envolvido com o balanço energético da planta. Para acrescentar novas informações relevantes sobre a função dos bZIPs do Grupo C e, a longo prazo, entender como a função de O2 evoluiu em angiospermas, iniciou-se neste trabalho uma caracterização detalhada da regulação dos membros do Grupo C em resposta a diversos sinais hormônais e a açúcares. Mostramos que apenas as hexoses glicose e manose e o ácido abscísico (ABA) regulam de maneira transiente a expressão dos genes bZIP do Grupo C, sugerindo que eles representam intermediários mediando as respostas a estes sinais. A glicose reprime a expressão de AtbZIP9 e de AtbZIP63 e induz a expressão de AtbZIP25, ABA reprime a expressão de AtbZIP63 e manose reprime a expressão de AtbZIP25 e de AtbZIP63. Em Arabidopsis, a hexoquinase1 (HXK1) é um sensor da glicose que ativa a síntese e sensibilidade ao ABA para inibir o desenvolvimento da plântula em resposta a glicose. Reportamos aqui que as repressões em curto prazo de AtbZIP9 e AtbZIP63 por glicose e de AtbZIP25 e AtbZIP63 por manose estão mediadas por vias de sinalização independentes de HXK1 e envolvem elementos relacionados a ABA. AtbZIP25 apresenta uma indução por glicose dependente de ABI5 e repressão por manose dependente de ABA2 e ABI4. A repressão de AtbZIP63 por glicose envolve uma via dependente de ABA2 e de ABI5 que é reprimida por ABI4. Já a repressão de AtbZIP63 por manose e de AtbZIP9 por glicose estão inseridas em vias independentes de ABA2, ABI4 e ABI5. A dependência diferencial de ABI5 e de ABI4 na regulação por glicose e manose de AtbZIP25 e de AtbZIP63, permite inferir que ambas hexoses atuam através de vias de transdução distintas e enfatiza a importância de manose como sinal metabólico de regulação. Observou-se ainda que ação conjunta de ABA e glicose apresenta um efeito sinérgico na repressão de AtbZIP63, provavelmente refletindo regulações pós-transcricionais da expressão deste gene. Os dados sugerem que AtbZIP63 representa um importante nó da comunicação entre a sinalização por ABA (estresse abiótico) e por glicose (nível energético) permitindo adequar eficientemente a resposta a estresse abiótico que seja compatível com o estado energético da organismo. / Abstract: In the model eudicot organism A. thaliana (Arabidopsis), four genes encoding bZIP transcription regulatory factors that are homologous to the maize Opaque-2 (O2) locus were identified. O2 is a key regulator of the carbon to nitrogen balance and of the prolamine type storage proteins synthesis during seed development. The Arabidopsis genes, AtbZIP9, the two paralogues AtbZIP10 and AtbZIP25 and AtbZIP63, the most probable O2-ortholgue, together form group C bZIP genes. AtbZIP9 is likely to be involved in phloem development while AtbZIP10 is related to stress responses but is also required for the regulation of seed storage protein genes very much like AtbZIP25. Finally, AtbZIP63 seems to be involved in the control of the energetic balance. In order to get new and relevant information about the role of the group C bZIP genes and consequently obtain new insight into the evolution of the O2-related functions in angiosperms, we initiated a detailed characterization of the regulation of group C members in response to hormonal signals and sugars. We show here that two hexoses, glucose and mannose as well as abscisic acid (ABA) are the only signals that transiently modulated the expression of group C bZIP genes, suggesting they are players in the response induced by these signals. While glucose is shown to repress the expression of AtbZIP9 and AtbZIP63 and to induce AtbZIP25 expression ABA is able to repress the expression of AtbZIP63 and mannose represses the expression of AtbZIP25 and AtbZIP63. In Arabidopsis, hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. We report that the short term regulation of the expression of AtbZIP9, AtbZIP63 by glucose and the repression of AtbZIP25 and AtbZIP63 by mannose are HXK1-independent and for AtbZIP25 and AtbZIP63, these regulations partly rely on ABA synthesis. It also shown that the activation of AtbZIP25 expression by glucose relies on ABI5 while its repression by mannose appears to be ABA2- and ABI4-dependent. Glucose repression of AtbZIP63 expression seems to involve an ABA2- and ABI5-dependent pathway which is repressed by ABI4. We also reveal that the regulations of AtbZIP63 by mannose and of AtbZIP9 by glucose do not require ABA, ABI4 or ABI5. The differential dependence of glucose and manose-induced regulation of AtbZIP63 and AtbZIP25 expression for ABI5 and ABI4 indicates that both hexoses act through distinct transduction pathways and highlights the importance of mannose as a regulatory metabolite. A synergetic repression of AtbZIP63 by ABA and glucose, which possibly reflects a post-transciptional regulatory scheme of AtbZIP63 expression, was uncovered. Together, the data suggests that AtbZIP63 is a key nod of the ABA (abiotic stress) and glucose (energetic balance) crosstalk network allowing to efficiently adjust the response to abiotic stresses according to the energetic status of the organism. / Tese (outorado) - Universidade / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Photosynthetic plants have a remarkable ability to modify their metabolism and development according to ever changing environmental conditions. The root system displays continuous growth of the primary root and formation of lateral roots enabling efficient water and nutrient uptake and anchorage of the plant in soil. With regard to lateral roots, development is post-embryonic, originating from the pericycle of the primary root. Coordinated activity of several molecular signalling pathways controlled by the hormone auxin is important throughout all stages of lateral root development.At first, two adjacent Xylem Pole Pericycle (XPP) cells are activated and the nuclei of these cells migrate towards a common cell wall.This is followed by XPP cells acquiring volume thus swelling up.The XPP cells then undergo anticlinal cell division, followed by a series of periclinal and anticlinal divisions,leading to lateral root primordia.These break through the radial cell layers and emerge out the primary root. Although root system plasticity is well-described in response to environmental cues such as ion nutrition in the soil, little is known on how root development is shaped according to the endogenous energy status of the plant.In this study, we were able to connect limited perturbations in photosynthetic energy supply to lateral root development.We established two experimental systems – treatment with low light and unexpected darkness which led to short-term energy imbalance in the plant.These short perturbations administered, showed an increase in the emerged lateral root density and decrease in root hexose availability and activation of the low energy marker gene ASN1 (ASPARAGINE SYNTHETASE 1).Although not demonstrated, presumably, these disturbances in the plant energy homeo-stasis activates SnRK1 (SNF1 RELATED KINASE 1),an evolutionary conserved kinase mediat-ing metabolic and transcriptional responses towards low energy conditions. In A. thaliana, two catalytic α-subunits of this kinase (SnRK1.α1 and SnRK1.α2) are functionally active and form ternary complexes with the regulatory β- and γ- subunits. Whereas unexpected darkness results in an increase in emerged lateral root density, the snrk1.α1 loss-of-function mutant displayed decrease in emerged lateral root density. As this effect is not that pronounced in the snrk1.α2 loss-of-function mutant, the α1 catalytic subunit is important for the observed lateral root phenotype under short-term energy perturbations. Moreover, root expression patterns of SnRK1.α1:GFP supports a role of this catalytic subunit in lateral root development. Furthermore, the lateral root response during short-term perturbations requires the SnRK1 downstream transcriptional regulator bZIP63 (BASIC LEU-CINE ZIPPER 63), as demonstrated here by a loss-of-function approach. Phenotypic studies showed that in comparison to wild-type, bzip63 mutants displayed decreased lateral root density upon low-light and unexpected darkness conditions. Previous work has demonstrat-ed that SnRK1 directly phosphorylates bZIP63 at three serine residues. Alanine-exchange mutants of the SnRK1 dependent bZIP63 phosphorylation sites behave similarly to bzip63 loss-of-function mutants and do not display increased lateral root density upon short-term unexpected darkness. This data strongly supports an impact of SnRK1-bZIP63 signalling in mediating the observed lateral root density phenotype. Plants expressing a bZIP63:YFP fu-sion protein showed specific localization patterns in primary root and in all developmental stages of the lateral root. bzip63 loss-of-function mutant lines displayed reduced early stage lateral root initiation events under unexpected darkness as demonstrated by Differen-tial Interference Contrast microscopy (DIC) and the use of a GATA23 reporter line. This data supports a role of bZIP63 in early lateral root initiation. Next, by employing Chromatin Immunoprecitation (ChIP) sequencing, we were able to iden-tify global binding targets of bZIP63, including the auxin-regulated transcription factor (TF) ARF19 (AUXIN RESPONSE FACTOR 19), a well-described central regulator of lateral root development. Additional ChIP experiments confirmed direct binding of bZIP63 to an ARF19 promoter region harboring a G-Box cis-element, a well-established bZIP63 binding site. We also observed that short-term energy perturbation upon unexpected darkness induced tran-scription of ARF19, which was impaired in the bzip63 loss-of-function mutant. These results propose that bZIP63 mediates lateral root development under short-term energy perturba-tion via ARF19. In conclusion, this study provides a novel mechanistic link between energy homeostasis and plant development. By employing reverse genetics, confocal imaging and high-throughput sequencing strategies, we were able to propose a SnRK1-bZIP63-ARF19 signalling module in integrating energy signalling into lateral root developmental programs. / Photosynthestisch aktive Pflanzen haben die bemerkenswerte Fähigkeit, ihren Stoffwechsel und ihre Entwicklung an sich ständig ändernde Umweltbedingungen anzupassen. Das pflanz- liche Wurzelsystem weist ein kontinuierliches Primärwurzelwachstum und eine Ausbildung von Seitenwurzeln auf, wodurch eine effiziente Wasser- und Nährstoffaufnahme sowie die Verankerung der Pflanze im Boden ermöglicht werden. Die Entwicklung der Seitenwurzeln verläuft post-embryonal, ausgehend vom Perizykel der Primärwurzel. Die koordinierte Aktivi- tät mehrerer molekularer Signalwege, die durch das Hormon Auxin gesteuert werden, ist in allen Stadien der Seitenwurzelentwicklung wichtig. Bei diesem Prozess werden zunächst zwei benachbarte Xylem-Pol-Perizykel-Zellen (XPP) aktiviert, deren Zellkerne zu einer gemeinsa- men Zellwand migrieren. Daraufhin schwillt das Volumen der XPP-Zellen an, bevor sich diese zunächst antiklinal teilen. Durch sukzessive periklinale und antiklinale Teilungen entstehen so Seitenwurzel-Primordien. Diese durchbrechen die radialen Zellschichten und treten aus der Primärwurzel aus. Während die Plastizität des Wurzelsystems als Reaktion auf Umwelteinflüsse, wie z.B. die Ver- sorgung mit Ionen aus dem Boden, bereits umfassend erforscht wurde, so ist die Abhängigkeit der Wurzelentwicklung vom endogenen Energiezustand der Pflanze weitgehend unbekannt. In dieser Arbeit konnten wir geringfügige Störungen der photosynthetischen Energieversor- gung mit der Seitenwurzelentwicklung in Verbindung bringen. Pflanzen wurden Schwachlicht oder unerwarteter Dunkelheit ausgesetzt und damit ein kurzzeitiges Energieungleichgewicht erzeugt. Hierdurch zeigte sich eine Zunahme der Seitenwurzeldichte bei gleichzeitiger Ab- nahme der Verfügbarkeit von Hexosen in der Wurzel und Aktivierung des Energieverarmungs- Markergens ASN1 (ASPARAGIN-SYNTHETASE 1). Obwohl dieser Mechanismus noch nicht ge- klärt ist, aktiviert die Störung der pflanzlichen Energie-Homöostase vermutlich SnRK1 (SNF1 RELATED KINASE 1), eine evolutionär konservierte Kinase, die metabolische und transkriptio- nelle Reaktionen auf niederenergetische Bedingungen vermittelt. In Arabidopsis sind zwei ka- talytische α-Untereinheiten dieser Kinase (SnRK1.α1 und SnRK1.α2) funktionell aktiv und bil- den ternäre Komplexe mit den regulatorischen β- und γ-Untereinheiten. Während eine uner- wartete Dunkelheit zu einer Zunahme der Dichte der auswachsenden Seitenwurzeln führt, zeigte die Snrk1.α1 Funktionsverlustmutante den gegenteiligen Effekt. Da dieser Effekt in der Funktionsverlustmutante von snrk1.α2 weniger stark ausgeprägt ist, scheint die katalytische Untereinheit α1 für den beobachteten Seitenwurzel-Phänotyp unter kurzfristigen Energiestö- rungen eine wichtige Rolle zu spielen. Das Expressionsmuster von SnRK1.α1:GFP in der Wur- zel unterstützt die mögliche Rolle dieser katalytischen Untereinheit bei der Seitenwurzelent- wicklung weiter. Darüber hinaus erfordert die Seitenwurzelbildung während kurzfristiger Störung des pflanzli- chen Energiehaushalts den SnRK1-nachgeschalteten Transkriptionsregulator bZIP63 (BASIC LEUCINE ZIPPER 63). Phänotypische Studien zeigten, dass bzip63-Funktionsverlust-Mutanten im Vergleich zum Wildtyp nach der Kultivierung unter Schwachlicht oder nach unerwarteter Dunkelheit eine geringere Seitenwurzeldichte aufwiesen. Frühere Arbeiten haben gezeigt, dass SnRK1 bZIP63 direkt an drei Serinresten phosphoryliert. Alanin-Austauschmutanten der SnRK1-abhängigen bZIP63-Phosphorylierungsstellen verhielten sich ähnlich wie bzip63-Funk- tionsverlustmutanten und zeigten bei kurzzeitiger unerwarteter Dunkelheit keine erhöhte Seitenwurzeldichte. Diese Daten weisen deutlich auf einen Einfluss des SnRK1-bZIP63-Signal- wegs auf den beobachteten Seitenwurzeldichte Phänotyp hin. Pflanzen, die ein bZIP63:YFP- Fusionsprotein exprimieren, zeigten ein spezifisches bZIP63 Lokalisierungsmuster in der Pri- märwurzel, sowie in allen Entwicklungsstadien der Seitenwurzel. bzip63-Funktionsverlustmu- tantenlinien zeigten reduzierte Seitenwurzel- Initiationsereignisse bei unerwarteter Dunkel- heit, wie durch Differentialinterferenzkontrast-Mikroskopie (DIC) und der Verwendung einer GATA23-Reporterlinie nachgewiesen wurde. Diese Ergebnisse deuten auf eine Rolle von bZIP63 bei der frühen Seitenwurzel-Initiierung hin. Durch die Anwendung der Chromatin-Immunopräzipitation (ChIP)-Sequenzierungsmethode konnten wir daraufhin globale Bindungsziele von bZIP63 identifizieren, einschließlich des au- xinregulierten Transkriptionsfaktors ARF19 (AUXIN RESPONSE FACTOR 19), einem gut be- schriebenen zentralen Regulator der Seitenwurzelentwicklung. Zusätzliche ChIP-Experimente bestätigten die direkte Bindung von bZIP63 an eine ARF19-Promotorregion, die ein G-Box cis- Element, eine bekannte bZIP63-Bindungsstelle, beherbergt. Wir beobachteten auch, dass kurzfristige Energiestörungen bei unerwarteter Dunkelheit die Transkription von ARF19 indu- zierte, die in der bzip63-Funktionsverlustmutante beeinträchtigt war. Diese Ergebnisse legen nahe, dass bZIP63 die Seitenwurzelentwicklung unter kurzfristiger Energiestörung über ARF19 vermittelt. Zusammenfassend lässt sich sagen, dass diese Studie eine neuartige mechanistische Verbin- dung zwischen Energiehomöostase und Pflanzenentwicklung herstellt. Durch den Einsatz von reverser Genetik, konfokaler Mikroskopie und Hochdurchsatz-Sequenzierungsstrategien konnten wir einen SnRK1-bZIP63-ARF19-Signalweg zur Integration von Energiesignalen in Sei- tenwurzelentwicklungsprogramme aufdecken.

Pflanzen müssen sich während der Samenkeimung und Keimlingsentwicklung über eingelagerte Speicherstoffe heterotroph versorgen, bis sie, nach Etablierung ihres Photosyntheseapparats, einen autotrophen Lebensstil führen können. Diese Arbeit geht von der Hypothese aus, dass der evolutionär konservierten zentral-metabolischen Kinase Snf1-RELATED PROTEIN KINASE 1 (SnRK1) eine besondere Rolle bei der Mobilisierung von Speicherstoffen während der Keimlingsentwicklung zukommt. Während die Bedeutung von SnRK1 als zentraler Regulator katabolischer Prozesse unter Energiemangel- und Stresssituationen bereits gezeigt wurde, war die Funktion von SnRK1 im Zusammenhang mit der Samenkeimung weitgehend ungeklärt. In dieser Arbeit konnte erstmals gezeigt werden, dass SnRK1 in Arabidopsis die Mobilisierung und Degradation von Speicherstoffen, insbesondere von Triacylglyceride (TAGs), Samenspeicherproteinen und Aminosäuren, steuert. Sowohl Studien zur Lokalisation von SnRK1:GFP-Fusionsproteinen als auch Kinaseaktivitätsassays unterstützen eine mögliche Funktion von SnRK1 während der Keimlingsentwicklung. Eine induzierbare snrk1-knockdown Mutante zeigt neben einem eingeschränkten Wurzel- und Hypokotylwachstum auch keine Ausbildung eines Photosyntheseapparats, was die zentrale Rolle der SnRK1 in diesem frühen Entwicklungsstadium untermauert. Durch Fütterungsexperimente mit Glukose konnte der Phänotyp einer snrk1 -Mutante in Keimlingen gerettet werden. Dies zeigt, dass der metabolische Block durch externe Gabe von Kohlenhydraten umgangen werden kann. Die zentrale Funktion von SnRK1 ist folgich der Abbau von Speicherstoffen und keine allgemeine Deregulation des pflanzlichen Stoffwechsels. Durch massenspektrometrische Untersuchungen von Keimlingen des Wildtyps und der snrk1-Mutante konnte gezeigt werden, dass TAGs in der Mutante in der spä- ten Keimlingsentwicklung ab Tag 4 langsamer abgebaut werden als im Wildtyp. Ebenso werden Samenspeicherproteine in der Mutante langsamer degradiert, wodurch die Verfügbarkeit von freien Aminosäuren in geringer ist. Entgegen der allgemeinen Annahme konnte gezeigt werden, dass während der Keimlingsentwicklung zumindest in Arabidopsis, einer ölhaltigen Pflanze, zunächst Kohlenhydrate in Form von Saccharose abgebaut werden, bevor die Degradation von TAGs und Aminosäuren beginnt. Diese Abbauprodukte können dann der Glukoneogenese zugeführt werden um daraus Glukose herzustellen. Mittels Transkriptom-Analysen konnten zentrale SnRK1-abhängige Gene in der Speicherstoffmobilisierung von TAG, beispielsweise PEROXISOMAL NAD-MALATE DEHYDROGENASE 2 (PMDH2) und ACYL-CoA-OXIDASE 4 (ACX4), und Aminosäuren identifiziert werden. Somit wurde ein Mechanismus der SnRK1-abhängigen Genregulation während der Samenkeimung in Arabidopsis gefunden. Bei der Degradation von Aminosäuren wird die cytosolische PYRUVATE ORTHOPHOSPHATE DIKINASE (cyPPDK), ein Schlüsselenzym beim Abbau bestimmter Aminosäuren und bei der Glukoneogenese, SnRK1-abhängig transkriptionell reguliert. Durch Koregulation konnte der Transkriptionsfaktor bZIP63 (BASIC LEUCINE ZIPPER 63) gefunden werden, dessen Transkription ebenfalls SnRK1-abhängig reguliert wird. Außerdem konnte die Transkription von cyPPDK in bzip63-Mutanten nur noch sehr schwach induziert werden. In Protoplasten konnte der cyPPDK-Promotor durch Aktivierungsexperimente mit bZIP63 und SnRK1α1 induziert werden. Durch Mutationskartierung und Chromatin-Immunopräzipitation (ChIP)PCR konnte mehrfach eine direkte Bindung von bZIP63 an den cyPPDK-Promotor nachgewiesen werden. Zusammenfassend ergibt sich ein mechanistisches Arbeitsmodell, in dem bZIP63 durch SnRK1 phosphoryliert wird und durch Bindung an regulatorische G-Box cis-Elemente im cyPPDK- Promotor dessen Transkription anschaltet. Infolgedessen werden Aminosäuren abgebaut und wird über die Glukoneogenese Glukose aufgebaut. Dieser Mechanismus ist essentiell für die Übergangsphase zwischen heterotropher und autotropher Lebensweise, und trägt dazu bei, die im Samen vorhandenen Ressourcen dem Keimling zum idealen Zeitpunkt zugänglich zu machen. Darüber hinaus werden Gene im Abbau von verzweigtkettigen Aminosäuren ebenfalls durch bZIP63 reguliert. Dabei wird dem Keimling Energie in Form von Adenosin-Triphosphat (ATP) zur Verfügung gestellt. Zusammengefasst zeigen die Ergebnisse dieser Arbeit, dass die Mobilisierung von Speicherstoffen auch während der Keimlingsentwicklung direkt von SnRK1 abhängig ist. Die umfangreichen Datensätze der RNA-Seq-Analysen bieten zudem die Möglichkeit, weitere SnRK1-abhängige Gene der Speichermobilisierung zu identifizieren und somit einem besseren Verständnis der Keimlingsentwicklung beizutragen. Aufgrund der zentralen Bedeutung der SnRK1-Kinase in diesem entscheidenden Entwicklungsschritt ist davon auszugehen, dass diese Erkenntnisse mittelfristig auch für bessere Keimungsraten und somit bessere Erträge in der Landwirtschaft genutzt werden können. / During seed germination and seedling establishment, seedlings must live heterotrophically on the resources stored in the seed. Only after establishing a fully functional photosynthetic apparatus, the young plant can change to an autotrophic lifestyle - one of the key features of plant life and metabolism. This work is based on the hypothesis that the evolutionarily conserved central metabolic kinase Snf1-RELATED PROTEIN KINASE 1 (SnRK1) plays a crucial role in the mobilization of storage compounds during seedling establishment. Whereas the importance of SnRK1 as a central regulator of catabolic processes during energy deprication and stress situations has already been demonstrated, so far, the function of SnRK1 in connection with seed germination had remained largely unresolved. Here, we shown for the first time that SnRK1 in Arabidopsis controls the degradation of storage resources, especially triacylglycerides (TAG), seed storage proteins and amino acids. Studies on the localization of SnRK1:GFP fusion proteins and as well as kinase activity assays support a possible function of SnRK1 during seedling establishment. An inducible snrk1-knockdown mutant is strongly impaired in root and hypocotyl growth and the plant do not develop a photosynthetic apparatus. Feeding experiments with glucose rescued the snrk1-mutant phenotype, showing that the metabolic block can be bypassed by external administration of carbohydrates. Thus, the central function of SnRK1 is concluded to be the degradation of the storage resources and rather than a general deregulation of the plant metabolism. Mass spectrometric investigations have shown that TAG degradation and seed storage proteins breakdown are partially impaired in the mutant. Thus, the availability of free amino acids in snrk1 mutant seedlings is lower in comparison to wildtype. It could be shown that - despite the fact that Arabidopsis is an oil-seed plant - carbohydrates, especially sucrose, are the primary resource compound for the seedling before the degradation of TAGs and amino acids is initiated. These degradation products can then be used in gluconeogenesis to produce glucose. Transcriptome analyses have identified key SnRK1-dependent genes, that play a key role in the storage resource catabolism, for example ACYL-CoA-OXIDASE 4 (ACX4) and PMDH2 in TAG breakdown. As an example of an enzyme involved in amino acid catabolism, cyPPDK was further investigated in the course of this study. During the degradation of amino acids, cyPPDK, a key enzyme in the degradation of certain amino acids and gluconeogenesis, is transcriptionally regulated in a SnRK1-dependent manner. Furthermore, it was discoverd that the SnRK1-dependent transcription factor bZIP63 (BASIC LEUCINE ZIPPER 63) is involved in the regulation of amino acid breakdown: In qRT-PCR experiments, bzip63-mutants showed no induction of cyPPDK, and co-regulation studies showed that cyPPDK and bZIP63 are subject to the same SnRK1-dependet regulation pattern during early seedling development. Finally, by mutation mapping and chromatin immunoprecipitation (ChIP)PCR, a direct binding of bZIP63 to the promoter could be demonstrated. Based on these results, a mechanistic working model was established, proposing bZIP63 to be phosphorylated by SnRK1 to then activate transcription of the cyPPDK promoter via binding to regulatory G-box cis elements. As a consequence, amino acids are degraded and the metabolites are used to produce glucose via gluconeogenesis. This additional source of energy enables the seedling to make the transition from heterotrophy to autotrophy. Additionally, the degradation of branched-chain amino acids is also regulated by bZIP63. Thereby, ATP is generated to fuel the seedlings energy demands. In summary, this study shows that SnRK1 plays an essential role in the mobilization of storage compounds in seedlings during the transition from heterotrophic to autotrophic life by supplying the seedling with the much-needed additional energy gained from the breakdown of TAGs and amino acids. Mining the extensive RNA-Seq data sets provided by this study will allow the identification of further SnRK1-dependent genes to further unravel this crucial signaling network. Due to the crucial role that these proteins play in early seedling development, these findings will enable future research to increase seedling vigor and finally crop yield.

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This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.