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.

Ebenso wie Tiere verfügen Pflanzen über die Fähigkeit elektrische Signale zu generieren. Dabei repräsentieren elektrische Signale – Membranpotentialänderungen an der Plasmamembran – die frühesten Antworten, welche an Pflanzenzellen im Zuge veränderter externer und intrinsischer Bedingungen beobachtet werden können. Stimuli wie Kälte, Hitze, Verwundung, Herbivorie und Pathogene, aber auch physiologische Prozesse, wie Wachstum und Bestäubung führen zur Änderung des Potentials der Plasmamembran pflanzlicher Zellen. Die meisten dieser Membranpotentialänderungen bestehen aus einer schnellen Depolarisation, gefolgt von einer Repolarisation des Membranpotentials, deren Kinetik, in Abhängigkeit des Stimulus hoch variabel sein kann. Das Wissen über die molekularen Grundlagen der Generierung und Weiterleitung elektrischer Signale in Pflanzen ist im Gegensatz zu Tieren nur wenig verstanden. Eine Ausnahme stellen ‚klassisch-erregbare‘ Pflanzen wie die Venusfliegenfalle oder die Mimose dar. In diesen Pflanzen führt ein Berührungsreiz zur Auslösung eines charakteristischen Aktionspotentials, welches in der Folge zu einer, auf differentiellen Turgoränderungen basierenden, nastischen Bewegung führt. In allen anderen Pflanzen ist die Kinetik der Membranpotentialänderungen sehr variabel, abhängig vom Stimulus und dem physiologischen Zustand der Zellen und – mit Ausnahme der Reaktion auf einen Kältestimulus – lediglich nach langen Latenzzeiten wiederholbar. Dieser Umstand verhindert eine systematische Analyse der molekularen Basis elektrischer Signale in den meisten Pflanzen. Ziel dieser Arbeit war es daher, auf der Basis des Channelrhodopsin-2 (ChR2) aus der Grünalge Chlamydomonas reinhardtii, welches bereits seit 2005 in der Neurobiologie genutzt wird, ein nicht-invasives Werkzeug zur funktionellen Analyse elektrischer Signale in Pflanzen zu etablieren. ChR2 ist ein Blaulicht-aktivierter Kationenkanal, der für seine Funktion all trans-Retinal als Cofaktor benötigt. Im Rahmen dieser Arbeit wurden verschiedene Varianten des ChR2, mit einem Schwerpunkt auf ChR2-C128T und vor allem ChR2-D156C, auch bekannt als ChR2-XXL eingesetzt. ChR2 konnte bereits durch M. Baumann im Rahmen ihrer Dissertation funktionell im transienten Expressionssystem Nicotiana benthamiana dargestellt werden. In der vorliegenden Arbeit wurde das System weiter ausgebaut und die besonders aussichtsreichen ChR2-Varianten nicht nur in N. benthamiana, sondern auch in stabilen Arabidopsis thaliana Linien funktionell charakterisiert. Dabei konnte mit dem ChR2-XXL ein geeignetes optogenetisches Werkzeug zur Untersuchung elektrischer Signale in Pflanzen identifiziert werden. ChR2-XXL bietet die Möglichkeit das Membranpotential durch kurze, 5 s Blaulichtpulse im Mittel um 95 mV zu depolarisieren und im Anschluss die Repolarisationsphase zu untersuchen. Blaulicht-induzierbare, ChR2-XXL-vermittelte Depolarisationen konnten, reproduzierbar und beliebig oft an den gleichen Zellen wiederholt ausgelöst werden. Dadurch ermöglicht ChR2-XXL die bisher nur unzureichend bekannten molekularen Komponenten der Repolarisation des Membranpotentials in Pflanzen zu erforschen. In tierischen Zellen generieren spannungsabhängige Natriumkanäle die Depolarisation, während spannungsabhängige Kaliumkanäle die Depolarisationskinetik bestimmen. Die im Vergleich zu tierischen Zellen veränderten Ionengradienten lassen vermuten, dass die pflanzliche Depolarisation im Wesentlichen durch Ca2+-abhängige Anionenkanäle vermittelt wird, die durch den Efflux von Cl- das Membranpotential depolarisieren. Für die Repolarisation wird zum einen die Beteiligung von auswärtsgleichrichtenden Kaliumkanälen postuliert. Zum anderen wird auch eine Beteiligung der Plasmamembran (PM) H+-ATPasen vermutet, welche gleichzeitig einen essentiellen Beitrag zur Generierung des Ruhepotentials leisten. In der vorliegenden Arbeit wurde es durch den Einsatz von ChR2-XXL möglich, beide potentiellen Komponenten der Repolarisationsphase, Kaliumkanäle und PM H+-ATPasen, erstmals durch eine nicht-invasive, Anionen-unabhängige Methode der Depolarisation zu untersuchen. Durch den Einsatz von Mutanten und Kaliumkanalinhibitoren konnte ein möglicher Beitrag des auswärtsgleichrichtenden Kaliumkanals Arabidopsis thaliana GUARD CELL OUTWARD RECTIFYING K+ CHANNEL (AtGORK) an der Repolarisationsphase in Arabidopsis Mesophyllzellen nahezu ausgeschlossen werden. Der auswärtsgleichrichtende Kaliumkanal GORK öffnet erst bei Membranpotentialen positiv vom Gleichgewichtspotential für Kaliumionen (EK (-118 mV)). Da die ChR2-induzierbaren Depolarisationen ebenso wie viele natürliche Stimuli, diesen Wert kaum erreichen oder nur geringfügig überschreiten, leistet der GORK einen geringfügigen Beitrag bei der Repolarisation. Dies ließ vermuten, dass die Repolarisation von EK bis zum Ruhepotential bei ca. -180 mV dagegen möglicherweise durch die PM H+-ATPasen bewerkstelligt wird. Die Wirkung des PM H+-ATPase Inhibitors Natriumorthovanadat, sowie des PM H+-ATPase Aktivators Fusicoccin auf die Repolarisationsphase konnten diese Hypothese unterstützen. Die Hemmung der PM H+-ATPasen verlangsamte die Repolarisationskinetik während eine Aktivierung der PM H+-ATPasen diese beschleunigte. So wurde es erstmals möglich den genauen Einfluss der PM H+-ATPasen auf Wiederherstellung des Membranpotentials während der Repolarisation in Mesophyllzellen zu studieren. Darüber hinaus wurde beobachtet, dass in Gegenwart des Kaliumkanalblockers Ba2+ die Repolarisation ebenfalls beschleunigt werden konnte. In Übereinstimmung mit dem ‚Pump-and-Leak‘-Modell (Alberts et al. 2002) deutet dies darauf hin, dass schwach einwärtsgleichrichtende Kaliumkanäle, wie der ARABIDOPSIS K+ TRANSPORTER 2 (AKT2) dem PM H+-ATPasen Protonengradienten entgegenwirken und somit das Ruhepotential aus der Summe der bewegten Ladungen von Pumpen und Kaliumkanälen bestimmt wird. Das mögliche Potenzial optogenetischer, Rhodopsin-basierter Werkzeuge für die molekulare Analyse elektrischer Signale, insbesondere unter Einsatz der breiten Palette lichtgesteuerter Pumpen und Kanäle, ihrer spektralen Diversität und ihrer Einkreuzung in ausgewählte Arabidopsis Mutanten wird diskutiert. / Like animals, plants have the ability to generate electrical signals – membrane potential changes on the plasma membrane. Electrical signals represent the earliest answers that can be observed in plant cells in the course of changing external and internal conditions. Stimuli such as cold, heat, wounding, herbivory and pathogens, as well as physiological processes such as growth and pollination, lead to changes in the potential of the plasma membrane of plant cells. Most of these membrane potential changes consist of rapid depolarization, followed by repolarization of the membrane potential, the kinetics of which can be highly variable depending on the stimulus. In contrast to animals, knowledge about the molecular basis of the generation and transmission of electrical signals in plants is poorly understood. Exceptions include "classically excitable plants" such as the Venus fly trap or mimosa, in which a touch stimulus leads to the triggering of a characteristic action potential, which subsequently leads to an elastic movement based on differential turgor changes. In all other plants, the kinetics of membrane potential changes are highly variable, depending on the stimulus and the physiological state of the cells and – with the exception of the reaction to a cold stimulus – can only be repeated after long latency periods. This prevents a systematic analysis of the molecular basis of electrical signals in most plants. The aim of this work was therefore to establish a non-invasive tool for the functional analysis of electrical signals in plants based on the channelrhodopsin-2 ChR2 from the green alga Chlamydomonas reinhardtii, which has been used in neurobiology since 2005. ChR2 is a blue light-activated cation channel that needed all-trans retinal as a cofactor in order to function. In this work, different variants of the ChR2, with a focus on C128T and especially D156C, also known as ChR2-XXL, were used. ChR2 could already be functionally represented by M. Baumann in the context of her dissertation in the transient expression system Nicotiana benthamiana. In the present work, the system was expanded and particularly promising ChR2 variants were characterized not only in N. benthamiana, but also in stable Arabidopsis thaliana lines. The ChR2-XXL identified a suitable optogenetic tool for examining electrical signals in plants. ChR2-XXL offers the possibility to depolarize the membrane potential by short, 5 s blue light pulses on average by 95 mV and then to investigate the repolarization phase. Blue light-inducible, ChR2-XXL-mediated depolarizations could be triggered reproducibly and repeatedly on the same cells as often as desired. ChR2-XXL enables research into the previously inadequate molecular components of the repolarization of the membrane potential in plants. In animal cells, voltage-dependent sodium channels generate depolarization, while voltage-dependent potassium channels determine the depolarization kinetics. The ion gradients changed compared to animal cells suggest that plant depolarization is essentially due to Ca2+-dependent anion channels, which depolarize the membrane potential through the efflux of Cl-. For repolarization, the involvement of outward rectifying potassium channels is postulated. On the other hand, participation of PM H+-ATPases is also suspected, which at the same time make an essential contribution to the generation of the resting potential. In the present work, the use of ChR2-XXL made it possible for the first time to investigate both potential components of the repolarization phase, potassium channels and PM H+-ATPases, using a non-invasive, anion-independent method of depolarization. Through the use of mutants and potassium channel inhibitors, a possible contribution of the outward rectifying potassium channel Arabidopsis thaliana GUARD CELL OUTWARD RECTIFYING K+ CHANNEL (GORK) to the repolarization phase in Arabidopsis mesophyll cells could almost be excluded. The outward-rectifying potassium channel GORK only opens at membrane potentials more positive than the equilibrium potential for potassium ions (EK (-118 mV)). Since the ChR2-inducible depolarizations, like many natural stimuli, hardly reach this value or only exceed it slightly, GORK makes a minor contribution to repolarization. This suggested that the repolarization from EK to the resting potential at approximately -180 mV, on the other hand, may be accomplished by PM H+-ATPases. The effects of the PM H+-ATPase inhibitor sodium orthovanadate and the PM H+-ATPase activator fusicoccin on the repolarization phase supported this hypothesis. The repolarization kinetics were slowed by inhibition of the PM H+-ATPases and accelerated by their activation. This made it possible for the first time to study the exact influence of the PM H+-ATPases on restoring the membrane potential during repolarization in mesophyll cells. It was also observed that repolarization could be accelerated in the presence of the potassium channel blocker Ba2+. In accordance with the 'pump-and-leak' model, this indicates that weakly inwardly-rectifying potassium channels, such as the Arabidopsis K+ Transporter 2 (AKT2) counteract the PM H+-ATPase-generated proton gradient and thus the resting potential from the sum of the moving charges of pumps and potassium channels is determined. The potential of optogenetic, rhodopsin-based tools for the molecular analysis of electrical signals, in particular using the wide range of light-controlled pumps and channels, their spectral diversity and their intersection with selected Arabidopsis mutants is discussed.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2016-2017 / Ces dernières années, l'intérêt pour l'accumulation de silicium (Si) chez les plantes a connu un essor considérable. Cet intérêt a été stimulé par la découverte des transporteurs d'influx et d'efflux de Si chez les plantes et par les nombreuses études qui rapportent les effets bénéfiques de son accumulation dans la lutte contre les stress. Récemment, la découverte d'un transporteur d'influx de Si chez le soya a permis de confirmer que l'espèce possède la capacité d'accumuler l'élément, alors qu'elle était initialement reconnue comme non-accumulatrice. L'identification subséquente de transporteurs d'efflux chez la même espèce (GmLsi2a, GmLsi2b, GmLsi2c) fait l'objet du présent mémoire. Le classement de 328 lignées de soya en neuf différents haplotypes selon le génotype rencontré chez les gènes GmLsi2b et GmLsi2c a permis de lier l'haplotype H1 à une accumulation accrue de Si chez le soya. Ce projet visait à vérifier si l'allèle spécifique de Lsi2 retrouvé chez de rares variétés de soya confère une absorption accrue de Si. Pour ce faire, nous avons utilisé un système hétérologue d'expression soit Arabidopsis thaliana, système déjà éprouvé pour le rôle de Lsi1 (Montpetit et al., 2012). L'accumulation de Si des différents allèles des gènes GmLsi2b et GmLsi2c ont été testés chez Arabidopsis. Les résultats obtenus suggèrent l'implication de l'haplotype H1 dans l'accumulation accrue de Si.

No description available.

The phosphatidylinositol signaling pathway utilizes inositol-containing second messengers to mediate signaling events. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. I have examined the substrate specificity of the At5PTase11 protein from the model plant, Arabidopsis thaliana. The At5PTase11 gene (At1g47510) encodes an active 5PTase enzyme that can dephosphorylate the phosphoinositide substrates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. In addition, the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways. To further delineate the function of At5PTase11 in Arabidopsis thaliana, two independent T-DNA insertion mutant lines were isolated (At5ptase11-1 and At5ptase11-2). Analysis of At5ptase11 mutant lines revealed that At5ptase11 mutant seeds germinate slower compared to wild-type seeds. Moreover, At5ptase11 mutant seedlings demonstrated less hypocotyl growth when grown in the dark. These results indicate that At5PTase11 is required for the early stages of seed germination and seedling growth. Since there are 15 predicted 5PTases in Arabidopsis thaliana, a group of 5PTases have been analyzed to identify the 5PTases with similar substrate selectivity. At5PTase1 (At1g34120), At5PTase2 (At4g18010) and At5PTase3 (At1g71710) have been found to hydrolyze all four potential substrates, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], PtdIns(4,5)P2, and PtdIns(3,4,5)P3. At5PTase7 (At2g32010) hydrolyzed PtdIns(4,5)P2, and PtdIns(3,4,5)P3 which is similar to the substrate selectivity of At5PTase11. In addition, At5PTase4 (At3g63240), and At5PTase9 (At2g01900) hydrolyzed only PtdIns(4,5)P2. These results indicate that there are different groups of Arabidopsis thaliana 5PTases based on the substrate selectivity. These results suggest that Arabidopsis thaliana 5PTases with similar substrate selectivity may have overlapping functions. In summary, the findings that At5PTase11 is a phospholipid-specific 5PTase and At5PTase11 functions in the early stages of seed germination and seedling growth indicate that 5PTases play important roles in plant growth and development. / Ph. D.

The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels. / Ph. D.

Plants and pathogenic microorganisms are in constant conflict with each other. Understanding the molecular networks that trigger resistance, along with the molecular networks that pathogens might co-opt to infect susceptible plants, is important for developing the integrated, holistic perspective that is necessary for innovative development of engineered resistance to current and emerging pathogens. The first objective of the dissertation was to increase the understanding of mechanisms by which plants recognize pathogen attack and mount an appropriate defense response. These experiments focused on resistance triggered by the Arabidopsis thaliana R gene, RPP7, which encodes a coiled-coil nucleotide binding-leucine-rich repeat (CC-NB-LRR) protein that activates race-specific resistance to the downy mildew pathogen, Hyaloperonospora parasitica (Hpa). Previously-published genetic epistasis tests have established that RPP7 activates defense responses through a signaling mechanism that does not require accumulation of salicylic acid (SA), or components of the ethylene and jasmonate response pathways. Furthermore, RPP7 is not strongly compromised by mutations in genes associated with defense signal transduction (PAD4, NDR1, NPR1, RAR1). Double mutant combinations of these signal transduction components were analyzed to detect additive or functionally-redundant contributions to RPP7-dependent resistance. Most of the double mutants support an enhanced level of asexual sporulation compared to the single mutant parental lines. Time-course experiments with histochemical stains revealed that these double mutants delay, but do not suppress, the oxidative burst and the hypersensitive response. These results suggest that RPP7 activates multiple signaling pathways, each of which makes incremental contributions to the timing of defense activation. The second objective of the dissertation was to investigate the role that auxin plays in enabling virulent H. parasitica to colonize Arabidopsis. Transcript profiling revealed induction of auxin-associated genes in response to infection of Arabidopsis thaliana by virulent strains of the oömycete pathogen, H. parasitica. Experiments with the DR5 / Ph. D.

The process of xylem tracheary element differentiation involves the coordination of vascular cambium activity, cell fate determination, cell expansion/elongation, secondary wall synthesis, programmed cell death, and cellular autolysis. The end result of tracheary element differentiation is a cellular corpse lacking a protoplast and consisting of a thickened cell wall composed mostly of lignin and cellulose. Little is known about the genetic mechanisms regulating the process of tracheary element differentiation. XCP2 expression localizes to tracheary elements according to two independent methods of analysis: promoter reporter experiments and immunogold localization by electron microscopy. XCP2 may be involved in catalyzing the degeneration of the protoplast during the final autolytic stages of tracheary element differentiation. To this date XCP2 function has not been directly demonstrated. In principle, any tracheary element-specific markers can be linked to upstream regulatory genes with roles in tracheary element differentiation. To develop the XCP2 promoter as a tool for identification of transacting factors, a promoter deletion analysis was carried out. Utilizing information from 5â and 3â deletion constructs, a 70-bp region upstream of the XCP2 translational start site is both necessary and sufficient for TE-specific expression of the UidA reporter gene. Mutational analysis of the ACTTTA element at position -113-bp strongly suggests it is a cis element required for XCP2 expression. In silico analysis of an 18-bp promoter region located within 200-bp of the translation start site and including the ACTTTA element revealed high indentity shared between xylem-specific XCP2 homologs from Zinnia elegans, Populus trichocarpa, and XCP1 from Arabidopsis thaliana. / Master of Science

Plants have evolved physical barriers and inducible defense responses to combat microbial pathogens. Inducible responses are mediated by R proteins, which recognize invading pathogens. R proteins must be precisely regulated to provide effective resistance, without inhibiting normal plant growth. However, little is known about R gene regulation under defense-inducing conditions. The interaction between the oomycete Hyaloperonospora parasitica and the model plant Arabidopsis thaliana provides an excellent model system to explore R gene regulation. My research focuses on RPP8, a CC-NBS-LRR gene, which provides resistance to the H. parasitica isolate Emco5. Previous work in the McDowell lab suggested that RPP8 is upregulated during defense responses. My research shows that RPP8 alleles from the Columbia and Landsberg erecta ecotypes are upregulated by H. parasitica and the defense signaling molecule salicylic acid, suggesting a potential feedback loop. RPP8-Ler is also systemically upregulated after infection of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Additionally, RPP8-Ler expression is increased during wounding and heat stress. I also examined the role of regulatory cis elements in the RPP8 promoter. Three W-boxes are essential for basal and inducible RPP8 expression, and are required for resistance to Emco5. The X-box, a unique cis element in the RPP8 promoter, is essential for strong basal expression and wound-induced upregulation, and affects spatial expression of RPP8-Ler. However, the X-box is not required for RPP8-Ler upregulation during pathogen or SA treatment. R genes may be induced as part of global defense responses, which could prime the host for more effective pathogen recognition. / Master of Science

Vitamin C (ascorbic acid) is one of the most essential organic compounds required by the human body for normal metabolic function. Unfortunately, this valuable nutrient is not produced in the human body but most plants and animal can produce this molecule. Although ascorbic acid was not isolated until the early part of the twentieth century, it was known that eating limes and other citrus fruits could ward off the affects of scurvy as early as the 1500's. Ascorbate serves many critical functions in plants as well as the human body. In both, it works as a cofactor in the production of hydroxyproline-rich compounds and helps protect molecules such as proteins, lipids and fatty acids from oxidation. Although the biochemical pathway in animals has been known since the 1950's (Jackel et al., 1950), the exact process by which ascorbic acid is made in plants has eluded scientists. It was shown in 1963 that the inversion of the hexose carbon chain, which occurs in the animal pathway, is not a possible mode of synthesis in plants (Loewus, 1963). As an alternative, a non-inversion pathway was proposed, which achieves ascorbic acid using D-mannose and L-galactose as intermediates, referred to as the Smirnoff-Wheeler pathway (Wheeler et al., 1998). It was shown that transforming lettuce (cv. Grand Rapids and Black Seeded Simpson) and tobacco (cv. Xanthi) with the terminal enzyme in the animal biosynthetic pathway (GLO; L-gulono-gamma-lactone oxidase) increases the ascorbic acid content between 4 and 7 fold. It was also shown through feeding studies that wild type tobacco plants had elevated ascorbate levels when fed the animal precursor (Jain and Nessler, 2000). These data suggest that at least part of the animal pathway could be present in plants, along with the Smirnoff-Wheeler (1998) pathway. To further investigate this discovery, wild type and ascorbic acid-deficient Arabidopsis thaliana were transformed with the glo. Homozygous lines of these transformants were generated and the ascorbic acid levels were compared to the untransformed wild type and mutant plants. Although the wild type plants containing glo did not show a significant increase in ascorbic acid production, all five of the vtc mutant lines had an increased ascorbic acid content relative to wild type level. These data suggest that an alternative pathway is present in plants that does not require many of the steps in the published Smirnoff-Wheeler (1998) pathway to produce ascorbic acid. / Master of Science