Rozprawy doktorskie na temat „Arabidopsis thaliana”

The shoot apical meristem (SAM) is the structure that shapes the aerial architecture of the plant, by producing lateral organs throughout development. In the model plant Arabidopsis thaliana, the SAM is always identifiable as a characteristic dome, whether it is found in the centre of a rosette of leaves or at the tip of an inflorescence. When senescence occurs and organogenesis ceases, the now inactive SAM still retains its characteristic appearance and it is never consumed into a terminal structure, such as a flower. Mutant plants that undergo termination represent a valuable tool to understand how the SAM structure and function are maintained during plant life. The aim of this work was to investigate the dynamics of meristem development through morphological and genetic studies of three Arabidopsis mutants that exhibit distinct modes of SAM termination: distorted architecture 1 (dar1), adenosine kinase 1 (adk1) and terminal flower 2 (tfl2). The dar1 mutation is characterised by a severely distorted cellular architecture within the SAM. We propose that dar1 affects the pattern of cell differentiation and/or cell proliferation within the SAM apical dome, resulting in termination by meristem consumption. Instead, the adk1 mutation affects the organogenic potential of the SAM, without altering its structure. The adk1 mutant has increased levels of cytokinins and, as a consequence of this, cell division is enhanced and cell differentiation is prevented in the apex, causing termination by meristem arrest. Finally, tfl2 is mutated in the conserved chromatin remodelling factor HP1, a transcriptional repressor with multiple roles during plant development. The tfl2 SAM terminates by conversion into a floral structure, due to de-repression of floral identity genes. Interestingly, tfl2 mutants also show an altered response to light, an indication that TFL2 might act as a repressor also in the context of light signalling.

In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

Die Plasmamembran lebender Zellen stellt die Hauptbarriere für alle Arten von extrazellulären Signalen dar. Viele davon werden ins Innere der Zelle weitergeleitet, hier lösen sie im Kern transkiptionelle Veränderungen und damit die Anpassung der Zelle auf Proteinebene aus. Andere wiederum werden direkt erkannt und in unmittelbare molekulare Antworten umgewandelt, wie zum Beispiel die Sekretion von gespeicherten Stoffen oder Konformations-änderungen von Proteinen. Besonders in Pflanzen, welche durch ihre sesshafte Lebensweise auf die rechtzeitige und spezifische Erkennung von Umweltveränderungen angewiesen sind, hat sich ein höchst diverses Rezeptorsystem entwickelt. In der Ackerschmalwand Arabidopsis thaliana, der in dieser Arbeit verwendeten Modellpflanze, wurden 610 verschiedene Rezeptorproteine identifiziert, welche wiederum von zahlreichen interagierenden, und bis jetzt weitestgehend unerforschten Proteinen reguliert werden. Als entscheidendes Prinzip, dieses Aufgebot an membran-gebundenen Komponenten von Signalkaskaden zu organisieren, gilt inzwischen die zeitliche und lokale Kompartimentierung der Plasmamembran. Durch Akkumulation relevanter Bestandteile von biologischen Prozessen in sogenannten Membrandomänen werden kurze Reaktionszeiten und die unmittelbare Signalweiterleitung garantiert. Besonders wichtig bei solchen Prozessen sind sogenannte Gerüstproteine, welche als Adaptoren zwischen anderen Komponenten fungieren. In dieser Arbeit wurden Remorine, eine Familie pflanzenspezifische Proteinen ohne bisher definierte Funktion, aufgrund ihrer Eigenschaft Membrandomänen zu markieren und ihrer mutmaßlichen Beteiligung an Pflanzen-Pathogen-Interaktionen, genauer untersucht. Eine systematische Expression von Remorinen als Fluorophor-Fusionen mit anschließender hochauflösender mikroskopischer und quantitativer Untersuchung offenbarte, dass die meisten Remorine sich in deutlich unterschiedlichen Mustern an der Membran verteilen. Untersucht wurden dabei Parameter wie die Größe der erkennbaren Domänen, die Form, die Helligkeit, aus welcher auf die Proteinkonzentration rückgeschlossen werden kann, sowie die Domänendichte an der Membran. Diese Ergebnisse wurden von Kolokalisationsanalysen unterstützt, welche die Lokalisation in unterschiedlichen, koexistierenden Membrankompartimenten erkennen ließen. Ferner wurden die Eigenschaften der von Remorinen markierten Membrandomänen, wie zum Beispiel der Austausch an Proteinen mit der umgebenden Membran, sowie lokale und zeitliche Dynamik und Stabilität untersucht. Dabei konnte eine hohe Fluktuation einzelner Proteine zwischen Domäne und umliegender Membran, jedoch eine klare laterale Immobilität der gesamten Domäne nachgewiesen werden. Zusätzlich zeichneten sich die untersuchten Domänen teilweise durch eine außerordentlich große zeitliche Stabilität aus, andere wiederum scheinen abhängig von bestimmten Stimuli zu entstehen. Weitergehende Arbeiten dienten der Identifizierung der Funktion einzelner Bereiche der Proteine. Hierbei konnte die entscheidende Rolle des äußersten C-terminalen Bereichs, des so- genannten RemCAs (Perraki et al., 2012; Konrad et al., 2014) als Membrananker bestätigt werden. Zusätzlich wurden mit Hilfe eines Hefe-2-Hybrid Ansatzes zahlreiche neue Interaktoren für eine Auswahl von Remorinen identifiziert. Dabei wurde ein essentieller Rezeptor der basalen Immunantwort, BAK1 als Interaktor für Remorin 6.4 gefunden. Zuletzt wurden einige wenige Remorine mit Hilfe von Mutantenlinien in einer genetischen Studie phänotypischen Analysen bezüglich ihrer Funktion bei Pflanzen-Pathogen Interaktionen unterzogen. Remorin 6.4 spielt hiernach eine Rolle bei der Immunantwort nach Befall mit virulenten Bakterien. Die grundlegende Erkenntnis, dass in lebenden Zellen zahlreiche klar unterscheidbare Arten an Membrandomänen koexistieren, ist ein Meilenstein auf dem Weg zur Anerkennung einer neuen Vorstellung vom Aufbau der Zytoplasmamembran. Diese wird häufig noch als undifferenzierte zweidimensionale Flüssigkeit beschrieben, in welcher stellenweise sogenannte Lipidflöße, festere Strukturen aus Cholesterin und Sphingolipiden, die auch bestimmte Proteine beherbergen können, auftreten. Anhand der in dieser Arbeit gewonnen Ergebnisse, sowie ähnlicher Studien in Hefe lässt sich nun folgendes Bild zeichnen: Es ist davon auszugehen, dass unterschiedliche Proteine, welche im selben biologischen Prozess involviert sind, in unmittelbarer Nachbarschaft oder sogar im selben Proteinkomplex in der Membran organisiert sind. Die Lipidzusammensetzung in der unmittelbaren Umgebung wird von diesen Proteinen bestimmt, bietet jedoch auch die Grundlage für die Bildung der Domäne, indem sie die Lokalisation der Komponenten in diesem Bereich fördert. Die zahlreichen an der Zellmembran gleichzeitig ablaufenden, unterschiedlichen Prozesse erfordern eine hochkomplexe, zeitlich und räumlich stark regulierte Kompartimentierung der Membran. Es kann vermutet werden, dass Remorine eine Rolle als Gerüstproteine bei der Ausbildung einer Auswahl dieser Domänen bilden. Im Fall von Remorin 6.4 ist das Protein für den Prozess der Flagellin-Erkennung und die unmittelbaren Abwehrantworten, welche nachweislich eine Präformierung der beteiligten Proteinkomplexe voraussetzen, notwendig.

Cyanide is highly toxic as it inhibits respiration in aerobic organisms by binding to cytochrome c oxidase in the mitochondrial electron transport chain. Plants naturally produce cyanide from the hydrolysis of cyanogenic glycosides and as a by-product of ethylene biosynthesis. β-Cyanoalanine synthase prevents self-poisoning by combining endogenous cyanide with cysteine in the mitochondria to form β-cyanoalanine, which is further hydrolysed to asparagine, or aspartate and ammonia, by plant nitrilase 4 enzymes. β-Cyanoalanine synthase activity enables plants to detoxify limited concentrations of exogenous cyanide. However, phytotoxicity and death occur from exposure to relatively low concentrations of exogenous cyanide. In contrast, some microorganisms have a high capacity for cyanide detoxification due to a number of metabolic pathways including the degradation of cyanide to formate and ammonia; or formamide, by bacterial cyanidase (CynD) and fungal cyanide hydratase (CHT), respectively. Environmental contamination caused by failure to contain cyanide from anthropogenic sources is an important global problem. Hydrometallurgical gold mining utilises cyanide as a lixiviant due to the high affinity of cyanide for gold and the stability of the resulting cyanometallic complexes in aqueous solution, and thus is a significant source of cyanide contamination of soil and water. Biological treatment methods for cyanide, such as phytoremediation, could provide alternatives to the currently used chemical destruction techniques with their associated disadvantages. The use of phytoremediation would require plants to tolerate high concentrations of cyanide in soil. Two attempts have previously been made, with some success, to increase cyanide tolerance in Arabidopsis by genetic engineering: the first, by augmenting the β-cyanoalanine synthase pathway using a microbial nitrilase; and, the second, by introducing a microbial detoxification pathway targeted to the chloroplasts while overexpressing the endogenous enzyme which metabolises the product of the cyanide detoxification reaction. The aim of the current study was to determine whether Arabidopsis thaliana could co-opt the CynD and CHT genes from the cyanide-degrading Bacillus pumilus and Neurospora crassa to detoxify higher levels of cyanide using the encoded enzymes, and whether targeting CynD and CHT to the mitochondria would confer a greater enhancement of cyanide tolerance on plants compared to targeting to the cytoplasm.

Ponts entre les séquences d'acides nucléiques et les protéines, les ribosomes sont des composants essentiels des cellules vivantes. Composé d'ARN et de protéines ribosomiques, ils sont transportés, durant leurs biogenèses, du nucléole au cytoplasme, où ils traduisent les ARN messagers (ARNm) en protéines. Ces dernières années, il a été montré que nombre de protéines ribosomiques étaient impliquées dans le développement d'Arabidopsis en intervenant sur la division et l'élongation cellulaire. L'impact d'un défaut de biogenèse des ribosomes sur le développement pourrait être expliqué par un effet dose, par une spécificité des ribosomes pour leur ARNm cibles ou par la multifonctionnalité de protéines ribosomiques. Les résultats obtenus montrent que REBELOTE (RBL), l'un des deux homologues chez Arabidopsis de la protéine NOC2p de levure, intervient probablement durant la biogenèse des ribosomes. Des mutations dans le gène RBL causent une gamme de phénotype de la létalité embryonnaire aux défauts de croissance (réduction de la taille de la plante, altération de la forme des feuilles...). Afin de mieux comprendre les processus contrôlés par RBL, la fonction ribosomique de RBL a été étudiée et ses interacteurs protéiques recherchés. Nous nous sommes ensuite focalisé sur les effets des mutations rbl sur la division et l'élongation cellulaire. Ce travail montre que les défauts observés aux niveaux moléculaire et cellulaire peuvent expliquer les retards de croissance des mutants rbl
Bridges between nucleic acids sequences and proteins, ribosomes are central components and the “auletes” of living cells. Composed of ribosomal proteins and RNA, they move during their biogenesis from the nucleolus to the cytoplasm, where they translate RNA messengers into proteins. In the past years, some mutants of ribosomal-biogenesis-related proteins have shown the importance of these proteins during cell division and Arabidopsis development. The impact of ribosomal defects on development could be explained by dose effect (which could be important for cell fitness), specificity of ribosomes for some mRNA or multifunctional ribosomal proteins (Mary E. Byrne, 2009). Here I present our work on REBELOTE (RBL), one of the two Arabidopsis homologs of the yeast NOC2 protein, which act during the ribosomal 60S subunit biogenesis. Mutations in REBELOTE gene cause a range of phenotypes, from embryo lethality to growth defects (reduced plant size, altered leaf shape…). To have a better understanding of RBL-controlled processes, we first analyzed the ribosomal function of RBL, and searched for its protein partners. Our results shows that RBL act in two different nucleolar complexes supposed to regulate 60S ribosomal subunit biogenesis. Subsequently, we focused on the effects of rbl mutations on the cell division/elongation processes. Our work shows that defects observed at molecular and cellular levels could explain the slow down of cell divisions and growth delay in rbl mutants

Nitrogen is often a limiting factor for plant growth due to its heterogenous distribution in the soil and to seasonal and diurnal changes in growth rates. In most soils, NH4+ and NO3 – are the predominant sources of inorganic nitrogen that are available for plant nutrition. In this context, plants have evolved mechanisms that enable them to optimize nitrogen acquisition, which include transporters specialized in the uptake of nitrogen and susceptible to a regulation that responds to nitrogen limiting or excess conditions. Although the average NH4+ concentrations of soils are generally 100 to 1000 times lower than those of NO3 – (Marschner, 1995), most plants preferentially take up NH4+ when both forms are present because unlike NO3–
NH4+ has not to be reduced prior to assimilation and thus requires less energy for assimilation (Bloom et al., 1992). Apart from high uptake rates in roots, high intracellular ammonium concentrations also result from quantitatively important internal breakdown of amino acids (Feng et al., 1998), and originates in high quantities during photorespiration (Mattson et al., 1997, Pearson et al., 1998). Thus, NH4+ is a key component of nitrogen metabolism for all plants and can accumulate to varying concentrations in all compartments of the cell, including the cytosol, the vacuole and in the apoplast (Wells and Miller, 2000; Nielsen and Schjoerring, 1998). Two related families of ammonium transporters (AMT1 and AMT2), containing six genes which encode transporter proteins that are specific for ammonium had been identified prior to this thesis and some genes had partially been characterised in Arabidopsis (Gazzarrini et al., 1999; Sohlenkamp et al. 2002; Kaiser et al., 2002). However, these studies were not sufficient to assign physiological functions to the individual transporters and AMT1.4 and AMT1.5 had not been studied prior to this thesis. Given this background, it was considered desirable to acquire a deeper knowledge of the physiological functions of the six Arabidopsis ammonium transporters. To this end, tissue specific expression profiles of the individual wildtype AtAMT genes were performed by quantitative real time PCR (qRT-PCR) and promoter-GUS expression. Modern approaches such as the use of T-DNA insertional mutants and RNAi hairpin constructs were employed to reduce the expression levels of AMT genes. Transcript levels were determined, and physiological, biochemical and developmental analysis such as growth tests on different media and 14C-MA and NH4+ uptake studies with the isolated insertional mutants and RNAi lines were performed to deepen the knowledge of the individual functions of the six AMTs in Arabidopsis. In addition, double mutants of the insertional mutants were created to investigate the extent in which homologous genes could compensate for lost transporter functions. The results described in this thesis show that the six AtAMT genes display a high degree of specifity in their tissue specific expression and are likely to play complementary roles in ammonium uptake into roots, in shoots, and in flowers. AtAMT1.1 is likely to be a ‘work horse’ for cellular ammonium transport and reassimilation. A major role is probably the recapture of photorespiratory NH3/NH4+ escaping from the cytosol. In roots, it is likely to transport NH4+ from the apoplast into cortical cells. AtAMT1.3 and AtAMT1.5 appear to be specialised in the acquisition of external NH4+ from the soil. Furthermore, AtAMT1.5 plays an additional role in the reassimilation of NH3/NH4+ released during the breakdown of storage proteins in the cotyledons of germinating seedlings. It was difficult to distinguish a specialisation between the transporters AtAMt1.2 and AtAMt1.1, however the root and flower specific expression patterns are different and indicate alternative functions of both. AtAMT1.4 has a very distinct expression which is restricted to the vascular bundels of leaves and to pollen only, where it is likely to be involved in the loading of NH4+ into the cells.The AtAMT2.1 expression pattern is confined to vascular bundels and meristematic active tissues in leaves where ammonium concentrations can reach very high levels. Additionally, the Vmax of AtAMT2 increases with increasing external pH, contrasting to AtAMT1.1. Thus, AtAMT2.1 it might be specialised in ammonium transport in ammonium rich environments, where the functions of other transporters are limited, enabling cells to take up NH4+ over a wide range of concentrations. The root hair expression ascribes an additional role in NH3/NH4+ acquisition where it possibly serves as a transporter that is able to acquire ammonium from basic soils where other transporters become less effective.RNAi lines showing a reduction in AtAMT gene mRNA levels and NH4+ transport kinetics, grew slower and flowering time was delayed. This indicates that NH4+ is a crucial and limiting factor for plant growth.
Ammonium stellt die von Pflanzen bevorzugte Aufnahmeform anorganischen Stickstoffes dar. Neben dem natürlichen Vorkommen im Boden, wird Ammonium während des Aminosäurestoffwechsels und der Photorespiration innerhalb des Pflanzenkörpers freigesetzt. Um Ammonium aus diesen Quellen zu assimlieren und zwischen den einzelnen Zellen zu transportieren hat die dikotyle Samenpflanze Arabidosis thaliana sechs veschiedene Ammoniumtransporter (AMT) evolviert. Zur Charakterisierung der spezifischen Funktionen der AMts wurden Expressionsprofile der jeweiligen Gene innerhalb der Wurzeln, des Sprosses und der Blüten unter verschiedenen Nährstoffbedingungen mittels quantitativer real-time PCR (qRT-PCT) und Promotor-GUS Expression erstellt. Weiter wurde die Fähigkeit zur spezifischen Regulation der einzelnen Transportergene in Abhängikeit des Stickstoffbedarfes der Pflanze analysiert. Zur Reduktion der mRNA Level der AMTs wurden RNA-Interferenz (RNAi) Mutanten erzeugt und um den Effekt des Verlustes einzelner AMTs zu studieren wurden T-DNA Insertionsmutanten dieser Gene isoliert. Von den mutanten Linien wurden die Transkriptionsraten durch qRT-PCR bestimmt und die NH4+ Transportleistungen durch 14C-Methylammonium (14C-MA) und Ammonium-Aufnahme Experimente analysiert. Weiter wurden Wachstumsanalysen der Mutanten auf verschiedenen Nährmedien durchgeführt und ihre vegetative und generative Entwicklung charakterisiert. Von den isolierten T-DNA Insertionsmutanten wurden Doppelmutanten aller Kombinationen hergestellt und phänotypisch analysiert.Es zeigte sich, daß der an der Plasmamembran lokalisierte AtAMT1.1 in allen Organen in hohen Konzentrationen exprimiert wird und unter Stickstoffmangelbedingungen vermehrt gebildet wird. Vermutlich dient er der generellen Assimilation von internen Ammonium und dessen Transport vom Apoplasten in den Symplasten. AtAMT1.3 und AtAMT1.5 sind in den Wurzeln und hier besonders in den epidermalen Wurzelhaaren exprimiert, die unter anderem der Aufnahme externer Nährsalze dienen. Beide unterliegen starker Regulation durch den internen Stickstoffbedarf der Pflanze. Es ist zu vermuten, daß sie primär der Aufnahme von Ammonium aus dem Boden dienen. AtAMT1.2 wird überwiegend an den vaskulären Geweben und den Blattparenchymzellen exprimiert. Überlappende Expressionsmuster mit AtAMT1.1 machen es schwierig eine spezifische Funktion zu identifizieren. AtAMT1.4 wird ausschließlich im männlichen Gametophyten exprimiert und dient hier vermutlich der Versorgung des Pollen mit Nährsalzen. AtAMT2 wird u.a. in den Wurzelhaaren exprimiert. Seine steigende Affinität zu Ammonium in basischem Milieu, umgekehrt zu AtAMT1.1, läßt vermuten, daß er der Pflanze die Ammoniumaufnahme bei steigendem pH ermöglicht. Im oberirdischen Sproß ist er überwiegend in Meristemen aktiv. Die in der Ammoniumaufnahme beeinträchtigten RNAi-Linien zeigten langsames Wachstum und blühten später als der Wildtyp. Es sind die ersten beschriebenen Ammoniumtransportermutanten, die einen solchen Phänotyp zeigen.

Alle Organismen sind für ihr Überleben auf Metalle angewiesen. Hierbei gibt es für jedes Metall einen Konzentrationsbereich, der das Optimum zwischen Metallmangel, -bedarf und -toxizität darstellt. Es gilt mittlerweile als erwiesen, dass alle Organismen zur Aufrechterhaltung des Metallgleichgewichts ein komplexes Netzwerk von Proteinen und niedermolekularen Verbindungen entwickelt haben. Die molekularen Komponenten dieses Netzwerks sind nur zu einem Teil bekannt und charakterisiert: In den letzten Jahren wurden einige Proteinfamilien identifiziert, deren Mitglieder Metalle durch Lipidmembranen transportieren. Eine dieser Metalltransporterfamilien ist die Cation Diffusion Facilitator (CDF)-Familie: Alle charakterisierten Mitglieder exportieren Metalle aus dem Zytoplasma – entweder in zelluläre Kompartimente oder aus der Zelle heraus. Von den zwölf Mitgliedern dieser Familie in Arabidopsis thaliana (A. thaliana) – Metall Toleranz Protein (MTP)-1 bis -12 – wurden bisher AtMTP1 und AtMTP3 charakterisiert. In dieser Arbeit wird die Charakterisierung von AtMTP2 beschrieben. Wie die homologen Proteine AtMTP1 und AtMTP3 führt AtMTP2 zu Zn-Toleranz, wenn es heterolog in Zn-sensitiven Hefemutanten exprimiert wird. Mit AtMTP2 transformierte Hefemutanten zeigten darüber hinaus erhöhte Co-Toleranz. Expression von chimären AtMTP2/GFP Fusionsproteinen in Hefe, A.thaliana protoplasten und in stabil transformierten A.thalinana Planzenlinien deutet auf Lokalisation of AtMTP2 in Membranen des Endoplasmatischen Retikulums (ER) hin, wenn GFP an den C-Terminus von MTP2 fusioniert wird. Fusion of GFP an den N-Terminus von AtMTP2 führte zu Lokalisation in der vakuolären Membran, was wahrscheinlichsten auf Fehllokalisierung durch Maskierung eines ER-Retentionsmotivs (XXRR) am N-Terminus von AtMTP2 zurückgeht. Dies legt nahe, dass AtMTP2 die erwähnten Metalle in das Endomembransystem der Zelle transportieren kann. Eine gewebespezifische Lokalisierung wurde mit Pflanzen durchgeführt, die das β-Glucuronidase (GUS)-Reporterprotein bzw. chimäre Fusionsproteine aus EGFP und AtMTP2 unter Kontrolle des nativen pMTP2-Promotors exprimierten. Diese Experimente bestätigten zum einen, dass der pMTP2-Promotor nur unter Zn-Defizienz aktiv ist. GUS-Aktivität wurde unter diesen Bedingungen in zwei Zonen der Wurzelspitze beobachtet: in den isodiametrischen Zellen der meristematischen Zone und in der beginnenden Wurzelhaarzone. Darüber hinaus konnte gezeigt werden, dass die EGFP-Fusionsproteine unter Kontrolle des nativen pMTP2-Promotors nur in epidermalen Zellen exprimiert werden. Für eine homozygote Knockout- Linie, mtp2-S3, konnte bisher kein eindeutiger Phänotyp identifiziert werden. Auf Grundlage der bisher durchgeführten Charakterisierung von AtMTP2 erscheinen zwei Modelle der Funktion von AtMTP2 in der Pflanze möglich: AtMTP2 könnte essentiell für die Versorgung des ER mit Zn unter Zn-Mangelbedingungen sein. Hierfür spricht, dass AtMTP2 in jungen, teilungsaktiven und damit Zn-benötigenden Wurzelzonen exprimiert wird. Die auf die Epidermis beschränkte Lokalisation könnte bei diesem Modell auf die Möglichkeit der zwischenzellulären Zn-Verteilung innerhalb des ER über Desmotubules hindeuten. Alternativ könnte AtMTP2 eine Funktion bei der Detoxifizierung von Zn unter Zn-Schock Bedingungen haben: Es ist bekannt, dass unter Zn- Mangelbedingungen die Expression der zellulären Zn-Aufnahmesysteme hochreguliert wird. Wenn nun die Zn-Verfügbarkeit im Boden z. B durch eine pH-Änderung innerhalb kurzer Zeit stark ansteigt, besteht die Notwendigkeit der Entgiftung von Zn innerhalb der Zelle, bis der starke Einstrom von Zn ins Zytoplasma durch die Deaktivierung der Zn-Aufnahmesysteme und einer geringeren Expression in der Pflanze gedrosselt ist. Ein ähnlicher Mechanismus wurde in der Bäckerhefe S. cerevisae beschrieben, in der darüber hinaus ein Zn-Transporter verstärkt exprimiert wird, der Zn durch Transport in die Vakuole entgiften kann. Es ist durchaus möglich, dass in Arabidopsis AtMTP2 die Zn-Detoxifizierung unter diesen speziellen Bedingungen durch Zn-Transport in das ER oder die Vakuole vermittelt. Zur Identifikation weiterer Komponenten des Metallhomöostasenetzwerks sind verschiedene Ansätze denkbar. In dieser Arbeit wurde in Hefe ein heterologer Screen durchgeführt, um Interaktoren für vier Mitglieder der Arabidopsis-CDF-Familie zu identifizieren. Unter den 11 im Hefesystem bestätigten Kandidaten befindet sich mit AtSPL1 ein AtMTP1-Interaktionskandidat, der möglicherweise eine Rolle bei der Cu-,Zn-Homöostase spielt. Als wahrscheinliche AtMTP3-Interaktionskandidaten wurde die c”-Untereinheit der vakuolären H+-ATPase AtVHA identifiziert sowie mit AtNPSN13 ein Protein, das vermutlich eine Rolle bei Fusionen von Vesikeln mit Zielmembranen spielt. Ein anderer Ansatz zur Identifikation neuer Metallhomöostasegene ist die vergleichende Elementanalyse von natürlichen oder mutagenisierten Pflanzenpopulationen. Voraussetzung für diesen Ansatz ist die schnelle und genaue Analyse des Elementgehalts von Pflanzen. Eine etablierte Methode zur simultanen Bestimmung von bis zu 65 Elementen in einer Probe ist die Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Der limitierende Faktor für einen hohen Probendurchsatz ist die Notwendigkeit, Proben für die Analyse zu verflüssigen. Eine alternative Methode der Probenzuführung zum Analysegerät ist die elektrothermale Verdampfung (ETV) der Probe. Zur weitgehend automatisierten Analyse von Pflanzenmaterial mit minimiertem Arbeitsaufwand wurde eine Methode entwickelt, die auf der Kopplung der ETV mit der ICP OES basiert.
All organisms require for their survival essential metals. For each required metal exists an optimal concentration between metal deficiency and -toxicity. It has become evident that all organisms developed a complex network of proteins and low molecular compounds to maintain the equilibrium between all metals. Only few molecular components of this metal-homeostasis network are characterized in detail: A number of protein families whose members transport metals over the barrier of lipid-membranes have been identified during the last couple of years. One of those metal-transport families is the Cation Diffusion Facilitator (CDF) family. All characterized members export metals from the cytoplasm – either into cellular compartments or outside the cell. From the 12 Arabidopsis thaliana (A.thaliana) members – Metal Tolerance Protein (MTP)-1 to 1-2 – only MTP1 and MTP3 have been characterized yet. In this work, characterization of MTP2 is described. As was found for the homologous proteins AtMTP1 and AtMTP3, heterologous expression of AtMTP2 in Zn-sensitive yeast mutants leads to enhanced Zn-tolerance. Less pronounced, enhanced tolerance was also found for Co when AtMTP2 was expressed in Co sensitive yeast mutants. Expression of chimeric AtMTP2/GFP fusion proteins in yeast, A.thaliana protoplasts and in stably transformed A.thalinana plant lines indicated localization of MTP2 in membranes of the endoplasmic reticulum, when GFP was fused to the C-terminal end of MTP2. Fusion of GFP to the N-terminal end of MTP2 lead to vacuolar localization that is most likely explained as mistargeting due to masking of an ER retrieval motive (XXRR) found at the N-terminus of MTP2. This suggests that AtMTP2 mediates the transport of Zn and Co into the endomembrane system of the cell. Tissue specific localization was performed with plant lines expressing the β-Glucuronidase (GUS) reporter protein and with plant lines expressing chimeric fusions of GFP with AtMTP2 under control of the native pMTP2 promoter. Those experiments confirmed Affymetrix Genechip® data suggesting activity of the pMTP2 promoter only under Zn-deficiency. GUS activity was only found under Zn-deficiency in two zone of root tips – the meristematic zone, characterized by isodiamtric cells, and in the beginning differentiation zone, characterized by appearing root hairs. Confocal microscopy with plant lines expressing chimeric MTP2 /GFP fusions demonstrated that expression of AtMTP2 is restricted to epidermal cells. A phenotype for the homozygous mtp2-S3 knockout mutant could not be identified yet. Based on the data obtained as yet
two mode of action of AtMTP2 in planta seam likely: AtMTP2 could be essential for delivery of Zn to the ER under Zn-deficiency. This is supported by the fact, that AtMTP2 is active in young, dividing (and therefore Zn-requiring) zones of the root. The epidermal-restricted expression of AtMTP2 points towards a distribution of Zn in these root zones of Zn within desmotubules. Alternatively, AtMTP2 could have a Zn-detoxifying function under Zn-shock. It is known that in yeast under Zn-deficiency not only the expression of an Zn-uptake transporter is up-regulated, but also the expression of a vacuolar Zn-transporter. It mediates Zn-detoxification of surplus Zn that enters cells upon Zn-resupply before shut down of the Zn uptake system. AtMTP2 could exert this function when soil Zn-availability raises suddenly, for example due to rain after a drought. Different means/methods are perceivable to identify further components of the metal homeostasis network. In this work, a heterologuos screen was performed in yeast to identify interacting proteins for four members of the Arabidopsis CDF-family. Among 11 candidates identified and confirmed in the Split Ubiquitin System (SUS, a Yeast-2-Hybrid variant) is with AtSPL1 an AtMTP1 interaction candidate, which plays putatively a role in Zn,Cu homoestasis. The c” subunit of the vacuolar H+-ATPase AtVHA was found as likely AtMTP3-interaction candidate, as well as AtNPSN13, an protein that plays putatively a role in fusion of vesicles with target-membranes. Another method to identify new metal homeostasis genes is the comparative elemental analysis of natural and mutagenized plant populations. Prerequisite for this approach is the fast and accurate analysis of the elemental composition of plants. An established method for elemental analysis is Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). The limiting factor for high thoughput is the requirement for laborious wet digest of plant samples before analysis. An alternative mean of sample delivery to the ICP OES is electrothermal vaporization (ETV). For faster, less laborious analysis of plant material, a method based on the established coupling of ETV with ICP OES was developed, which is optimized for plant material and automated as far as possible.

Glycosyltransferases (GTs) constitute a large family of enzymes that are involved in the biosynthesis of a diversity of glycoconjugates. Family 1 GTs catalyse the transfer of sugars from activated sugar donors to a number of small lipophilic acceptor molecules. In the Arabidopsis genome, one hundred and seven GT-encoding genes from Family 1 have been identified by sequence analysis. The in vitro analysis of their gene products revealed that Arabidopsis thaliana (A. thaliana) GTs form glycosides of a number of plant secondary metabolites, phytohormones and of non-natural small molecules (xenobiotics) with high selectivity. When A. thaliana GTs were expressed in E. coli cells, their utility as whole-cell biocatalysts for the production of selective glycoconjugates has been demonstrated. This approach combines two advantages: first, the use of activated donor sugars for the reaction derived from the biosynthetic pool present in E. coli and second, recovery of the formed glycoside in the culture medium followed by classical purification methods. In this context, A. thaliana GTs may have the potential for development into biocatalysts for the synthesis of glycosides of pharmaceutical and nutraceutical importance. Therefore, a rapid screening method applicable for GTs active in whole-cell biocatalysis was developed such that one could scale-up rapidly from identification of a GT in a 96-well plate screen to use of the enzyme in a fermenter system for the synthesis of glycoconjugates (Chapter 3). The application of the whole-cell screen towards three aromatic substrates, trans-resveratrol, podophyllotoxin and daidzein, identified twenty-five and thirteen GTs with interesting selectivities that recognise trans-resveratrol and daidzein respectively (Chapter 4). Structural analyses of GTs revealed two structural superfamilies, named according to their fold GT-A or GT-B. Structures of five Family 1 GTs have been solved and shown to consist of the GT-B topology. A GT-B fold comprises two domains; the C-terminal domain responsible for donor sugar binding whereas the N-terminal binds the acceptor molecule. Since it is believed that within a family the same fold occurs, A. thaliana GTs should be a member of the GT-8 superfamily. Therefore, this study also investigated the role of either domain in sugar donor and acceptor recognition by exchanging the N-terminal domains between two similar A. thaliana GTs followed by a detailed kinetic analysis of the mutants (Chapter 5).

Programmed Cell Death (PCD) describes an orderly cellular breakdown that occurs in both plants and animals throughout development and in response to biotic and abiotic stresses. The molecular machinery that functions in the induction and execution of animal PCD has been characterised in great detail. Conversely, few genes and proteins involved in plant PCD have been identified. While certain features of animal PCD may be conserved, the induction and execution of plant PCD is also likely to involve novel proteins and mechanisms. The aim of the work presented in this thesis was to investigate experimental approaches for studying plant PCD and to gain an understanding of the molecular mechanisms involved. To this end, an Arabidopsis thaliana cell suspension system was developed in which PCD could be induced by both a heat treatment (55°C, 10 min) and senescence (13 to 14 days-old). This system allowed for the molecular responses related to programmed cell death to be distinguished from those that were a specific response to the inducing stimulus. The Arabidopsis cell suspension system was utilised for an analysis of transcriptomic and proteomic changes that occur following the induction of PCD. A custom cDNA microarray analysis of ~100 putative cell death-related genes was used to measure the abundance of transcripts of these genes during PCD, and this work was extended to a whole-genome transcriptomic analysis of PCD. A number of candidate genes that may play a role in plant PCD were identified. These included those encoding antioxidant enzymes, cytosolic heat shock proteins, the mitochondrial adenine nucleotide translocase, ion transporters, a two-component response regulator (ARR4), several pathogenesis-related proteins, phospholipases and proteases, extracellular glycoproteins and enzymes (including a subtilisin-like protease, chitinases, and glucanases), and transcriptional regulators such as a homeobox leucine zipper and NAC-domain proteins. The induction and execution of plant PCD is also likely to involve mechanisms that are not transcriptionally regulated. A proteomic analysis of changes in the total cellular protein profile during heat- and senescence-induced PCD was therefore used to identify 12 proteins that are modulated in both systems and may play a PCD-specific role. These included the mitochondrial voltage-dependent anion channel (Athsr2), catalase, mitochondrial superoxide dismutase, an extracellular glycoprotein, and aconitase. Selected genes and proteins identified in the transcriptomic and proteomic analyses were further investigated in an attempt to define their role in plant PCD. Since PCD is difficult to quantitatively analyse at the whole-plant level, initially a strategy of transient expression of genes of interest in Arabidopsis protoplasts was adopted. However, it proved to be technically difficult to accurately quantify the number of dead cells in this system. As an alternative, Arabidopsis T-DNA insertional mutants within genes of interest were investigated for PCD-related phenotypes. Mutants in Senescence-Related Gene 3, the mitochondrial voltage-dependent anion channel (Athsr2), and cytosolic Heat shock protein 70-3 were isolated. The mutant lines were not visibly affected in their development, formation of xylem, onset and progression of senescence, or responses to abiotic and biotic stresses. This indicated that these genes are either not involved in the PCD pathway or that their functional role can be fulfilled by other gene products.

Orientador: Michel Georges Albert Vincentz
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Genetica Vegetal e Melhoramento
Doutora em Genética e Biologia Molecular

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第13129号
農博第1634号
新制||農||942(附属図書館)
学位論文||H19||N4255(農学部図書室)
UT51-2007-H402
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 坂田 完三, 教授 梅澤 俊明, 教授 矢﨑 一史
学位規則第4条第1項該当

Gravitropism is an important tropic response in plants that allows the ability to respond to changes in orientation and to modify growth to maintain specific patterns of growth with respect to gravity. Gravitropism is controlled by a group of hormones called auxins. There are three steps that contribute to the response: the perception of the gravity signal, the differential flow of auxin, and the transcriptional control of auxin related genes. The so-called sine law of gravitropism states that the magnitude of a plants’ gravity response is proportional to the sine of the angle between the organ axis and the vertical. This model has since been described in many species, but the molecular basis of the mechanism remains unclear. Using kinetics experiments, auxin-mediated gravity response in Arabidopsis roots was confirmed to be angle-dependent. The auxin reporter R2D2 was used to quantify previously unreported angle-dependent auxin asymmetries that likely govern gravity response in primary and lateral roots. The subcellular localisation of auxin efflux carriers, known as PINs, were quantified in the gravity-sensing cells of primary roots. It was found that as the angle of stimulation increases, PINs are targeted more to the basal plasma membrane. Evidence for angle-specific contributions of PINs was also found. Other components of gravity response were also considered. The role of auxin signalling in the gravity-perception and response in Arabidopsis roots and shoots was investigated using gravity-sensing cell- and epidermal cell-specific promoters expressing mutated versions of Aux/IAA and ARF auxin signalling regulators. Gravitropism assays with these transgenic lines indicate that auxin signalling is necessary in the gravity-sensing cells of the primary root, however, its exact role is still unclear.

Ribosomes serve as the site of protein synthesis in all living cells. Ribosomes were discovered in 1955 by George E. Palade when he was studying the endoplasmic reticulum which is covered by ribosomes. He received the Nobel Prize in Physiology or Medicine in 1974 for this discovery. Ribosomes are large protein and rRNA complexes which are made up from one small and one large subunit that work together to translate mRNA into a protein chain. Eukaryotic translation is mainly controlled during the initiation, which involves protein phosphorylation. In plants there is a general increase of protein synthesis during the day in order to synthesize proteins needed for photosynthesis. Phosphorylation can alter protein function and localization and is reversibly added and removed by kinases and phosphatases, respectively. The aim of the studies in this thesis was to elucidate the phosphorylation status of ribosomal proteins in the Arabidopsis thaliana 80S ribosome. I have focused on comparing ribosomal protein phosphorylation between different conditions and sub cellular locations, namely day/night conditions and cytosol/nucleus location. By using Fe3+IMAC to enrich phosphorylated peptides from cytosolic ribosomes followed by mass spectrometric analysis eight serine residues in six ribosomal proteins were found to be phosphorylated. Among these was a novel phosphorylation site in 40S ribosomal protein S6 at Serine 231. By using quantification with stable isotope labeling and mass spectrometry this phosphorylated residue and three other ribosomal phosphopeptides were found to have increased phosphorylation levels during day as compared to night ranging from 2 to 4 times. This phosphorylation increase can in turn effect the modulation of the diurnal protein synthesis in Arabidopis thaliana. Ribosome biogenesis involves shuttling of proteins and ribosomal subunits between the cell nucleus and cytoplasm. By purifying ribosomal proteins from these two cellular compartments and enriching for phosphopeptides using TiO2 affinity chromatography combined with mass spectrometry I was able to analyze their phosphorylation status. This method identified 13 phosphopeptides derived from 11 ribosomal proteins as well as phosphopeptides from two ribosomal associated proteins. 40S ribosomal protein S2-3 was found phosphorylated only in the cytoplasmic samples while 60S ribosomal protein L13-1 and the two ribosomal associated proteins were found only in the nuclear enriched samples.

Bibliography: leaves 89-104. In this study an EMS-mutated Arabidopsis mutant pho2, which accumulates Pi in leaves, was used to study Pi uptake and transport by comparing it to wild-type seedlings. The study aimed to define the physiological lesions in pho2 mutant and to obtain evidence regarding the function of the PHO2 gene in P nutrition in higher plants. Accumulation of Pi in leaves of pho2 was found to reside in the symplast and was not related to Zn-deficiency. The physiology of the pho2 mutant is consistent with either a block in Pi transport in phloem from shoots to roots or an inability of shoot cells to regulate internal Pi concentration. Southern block analysis revealed that the two transporter genes, APT1 and APT2 were not responsible for the pho2 mutant. Data from the mapping of the PHO2 gene along with information from the Arabidopsis genome sequencing will form the basis for cloning the PHO2 gene in the future.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is a phosphatase dually targeted to both chloroplasts and mitochondria. Overexpression (OE) of AtPAP2 in Arabidopsis thaliana was reported to speed up plant growth and promote flowering, seed yield and biomass at maturity in a previous study. Under long-day (16 hours light at 22°C / 8 hours dark at 18°C) growth conditions, the leaves of 20-day-old OE lines contained significant higher sucrose and glucose than the wild-type (WT) plants, reflecting their high energy status. The AtPAP2 OE line is thus a good model to investigate the impact of high energy on the global physiological changes in Arabidopsis. In this study, the systems biology of the high-energy plants, in terms of transcriptome, proteome, metabolome and small RNA (sRNA) expression profiles were examined. Liquid chromatography mass spectrophotometry (LC-MS/MS) and enzyme assays were both employed to measure the content of ADP/ATP/NAD/NADP/NADPH in the leaves of 20-day-old OE and WT lines at 3 time points (t = 0, end of night; t = 1, 1 h after light was on; t = 8, 8 h after light was on). My results showed that the ATP and ADP contents were significantly increased in the OE line at all the three time points. In the dark, since mitochondria is the major source of ATP supply in plant cells, the comparison of omics data between the OE and WT lines at t = 0 reflected the impact of high ATP output from mitochondria on plant leaves. Similarly, the comparison between the OE and WT lines at t = 8 reflected the impact of higher ATP output from chloroplast of plant leaves. By RNA sequencing (RNA-Seq) technology, transcriptome profiles between OE and WT at all the three time points (t = 0, 1, and 8 h after illumination) were compared. The transcripts of 29,435 genes were detected in all six datasets. Moreover, transcripts encoded by the chloroplast and mitochondrial genomes, which were not reported in the previous microarray studies were also sequenced. Genes of the central energy conversion pathways that were differentially transcribed between the OE and WT lines were identified. In proteomics studies, 2663 proteins were identified by the isobaric tags for relative and absolute quantification (iTRAQ) labeled technique. The proteins with significantly altered in protein abundances (P < 0.05) in various energy pathways, including photosynthesis, redox regulation, Calvin-Benson cycle, carbohydrate metabolism, glycolysis, tricarboxylic acid (TCA) cycle and the respiratory system in mitochondria, were identified. When the transcriptomes and proteomes were compared, the correlation between mRNA transcript and protein abundances was not high. Small RNA studies showed that the expression of miR173, which initiates the generation of tasiRNAs from TAS1 and TAS2 loci, was significantly increased in the leaves of OE lines. These tasiRNAs target the mRNAs of various pentatricopeptide repeat (PPR) and tetratricopeptide repeat (TPR) proteins, which control RNA metabolism in chloroplasts and mitochondria. A network of miR173-tasiRNAs-PPR/TPR was established to present the changes in the OE lines. In summary, my study systematically investigated the global impact of high energy status to the metabolome, transcriptome, proteome and sRNAs profiles of Arabidopsis. Two diagrams were presented to illustrate the impacts of high energy status on the physiology of leaf cells under light and dark conditions, respectively.
published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy

Roots are essential for plant anchorage and nutrient uptake. Although root system architecture is genetically determined, its high level of plasticity allows rapid as well as long term adaptation to the plants' environment. To confer such adaptability, favorable alleles have been selected within species to endow them with an optimized root system. The identification of such favorable alleles is therefore a central component of research in plant breeding.
In an attempt to isolate novel factors that modulate root development, we exploited natural genetic variation in the model plant Arabidopsis thaliana. Tissue culture analysis of 44 accessions led to the identification of a line, Uk-1 (Umkirch-1), whose root system differs significantly from average accessions. A short primary root and an increased number of lateral and adventitious roots are typical for the Uk-1 root system, while the average Arabidopsis root system consists of a predominant primary root and few lateral roots. The major locus responsible for the Uk-1 phenotype, BREVIS RADIX (BRX), was successfully isolated by map-based cloning. Quantitative trait locus (QTL) analysis revealed that BRX is responsible for ca. 80% of the variance of the observed primary root length difference as compared to an average control accession.
BRX controls the extent of cell proliferation and elongation in the growth zone of the root tip and is a member of a novel, small family of proteins that contain three distinct and highly conserved domains of unknown function. BRX is a low abundant, nuclear protein, which is expressed in the phloem and the pericycle at the phloem poles. BRX is also expressed in the columella. Since BRX is not expressed in the root meristem, it must affect root growth in a secondary manner, likely by modulating responses to the plant hormone auxin.
Transcriptional profiling in root tissues revealed that BRX does so by maintaining optimal expression of the brassinosteroid biosynthesis gene CPD, and thereby optimal endogenous levels of the plant hormone brassinosteroid. Further analysis identified BRX as a central component of the interdependency of brassinosteroid signaling and auxin signaling.

The model plant Arabidopsis thaliana has been very successful thus far as a tool for understanding and studying the genetics of plant development. Analysis of its sequenced genome revealed the occurrence of duplicated chromosome blocks, resulting in duplicated genes. Duplicated genes, high in sequence and/or structure similarity, form gene families. One such family, of BRX-like genes, is presented in this thesis. All members contain a characteristic "BRX" domain that is required for BRX activity in planta. BRX (BREVIS RADIX), is a novel regulator of root growth in Arabidopsis. However, analyses of Arabidopsis single and double mutants with other gene family members, suggests that BRX is the only member with a dominating role in root development. Interestingly, BRXL1, although having BRX activity in the root, does not act redundantly with BRX in vivo, presumably because it is expressed at much lower levels than BRX. These two gene family members demonstrate the uncommon phenomenon of unequal genetic redundancy in plants. Another gene family member, BRXL4, although non-redundant with regards to BRX activity in the root, did display novel shoot-related phenotypes when over-expressed. In these lines the lateral shoots and hypocotyls showed increased Gravitropic Set-Point Angles resulting in the downward growth of the adult lateral shoots and a wide range of growth directions in the hypocotyls. Over-expression lines of BRXL4 also displayed seedling agravitropism. Auxin-induced transcription as monitored by the DR5::GUS reporter, is altered in these lines compared to wild-type. Additionally, hypocotyl curvature, stem bending and amyloplast localization profiles in response to a change in gravity vector, are also altered in these over-expression lines and in the brxl4 mutant compared to wild-type.

Les tissus vasculaires sont d’une grande importance pour la physiologie et le développement de la plante. Le xylème et le phloème composent ce tissu. En assurant le transport des sèves brutes et élaborées, ils permettent ainsi de redistribuer minéraux et métabolites entre les différents organes de la plante. La régulation de la mise en place de ces deux tissus est hautement contrôlée tant d’un point de vue génétique que spatio-temporel. Parmi les gènes impliqués dans la mise en place des tissus vasculaires, le gène OCTOPUS (OPS) intervient très précocement au cours du développement dans la mise en place du patron vasculaire ainsi que différenciation du phloème(Bauby et al., 2007; Truernit et al., 2012). OPS appartient à une famille de gènes spécifiques des plantes supérieures. Cependant la fonction moléculaire de la protéine OPS reste inconnue. Une combinaison d’approches physiologique, biochimique, moléculaire, cytologique et génétique a permis de montrer que le gène OPS est un nouveau régulateur positif de la voie signalisation des brassinostéroïdes (BR). Les BR sont des hormones végétales impliquées dans de nombreux processus développementaux incluant l’élongation cellulaire ou encore l’organisation des tissus vasculaires. Bien que la découverte des BR date seulement de 1970 (Mitchell et al., 1970; Mitchell and Gregory,1972; Grove et al., 1979), l’intérêt commun que les scientifiques lui ont porté fait que sa voie de signalisation est aujourd’hui très détaillée. Parmi les composants de la voie des BR, la protéine kinase BIN2 en est le régulateur clef (Li et al., 2001) réprimant la voie par son action sur les facteurs de transcription BES1 et BZR1(Yin et al., 2002; Wang et al., 2002). OPS interagit physiquement avec la protéine BIN2 au niveau de la membrane plasmique, pouvant créer une inhibition par délocalisation de la protéine BIN2 de son site d’activité. A ce titre, les défauts de phloème observés chez le mutant opssont restaurés lorsque l’activité de BIN2 est abolie ou lorsque la voie est induite en dessous de BIN2. Ainsi nous montrons que la voie des BR est directement impliquée dans la différenciation du phloème. Plus généralement, une étude de la redondance fonctionnelle d’autres membres de la famille OPS semble indiquer qu’ils pourraient exercer une fonction similaire de régulateurs positifs de la voie des BR au sein d’autres tissus de la plante
Vascular tissues play an important role in plant physiology and development. Vascular tissues consist on xylem and phloem that ensure sap transport and permit to redistribute mineral and metabolites between different organs. Regulation of vascular tissues establishment is highly controlled in space and time. Among genes involved in vascular tissues formation, OCTOPUS (OPS) gene operates early during development to control vascular patterning and to induce phloem differentiation(Bauby et al., 2007; Truernit et al., 2012). OPS belongs to a multigenic family conserved in high plant. However, molecular function of OPS protein remains unclear. A combination of physiological, biochemical, molecular, cytological and genetic approaches allowed to show that OPS gene is a new positive regulator of brassinosteroid (BR) signaling pathway. BR are phytohormones involved in many developmental processes such as cell elongation or vascular tissues organization. Although BR were discovered in 1970 (Mitchell et al., 1970; Mitchell and Gregory, 1972; Grove et al., 1979), interest of researchers for this hormone permitted its detailed description. Among the component of BR signaling, the BIN2 kinase is the key regulator (Li et al., 2001) which represses the pathway through its action on BES1 and BZR1 transcription factors(Yin et al., 2002; Wang et al., 2002). OPS interacts physically with BIN2 at the plasma membrane which could create an inhibition by delocalization of the BIN2 protein from its activity place. As such, opsphloem defects are restored when the activity of BIN2 is inhibited or when the pathway is induced downstream of BIN2. Thus, we show that BR pathway is directly involved in phloem differentiation. More generally, a study of the functional redundancy of other OPS family members suggests that they could have a similar function as positive regulators of BR pathway in other plant tissues

Les stress abiotiques (températures extrêmes, salinité, sécheresse) ont un impact négatif sur la croissance et le développement végétal et constituent un défi agronomique majeur. La compréhension des réponses mises en œuvre par différentes espèces de plantes est un volet incontournable de toute stratégie d'amélioration variétale, mais offre aussi un modèle de choix pour l'étude intégrative de mécanismes qui opèrent à différents niveaux d'organisation biologique, selon des modalités hautement régulées. L'abondance de collections de génotypes et le progrès des outils d'analyse moléculaires, couplés à des traitements bioinformatiques performants, ont ouvert la perspective de caractériser la plasticité phénotypique de plantes modèles à l'échelle génomique et dans un contexte environnemental dynamique. La métabolomique en particulier, définie comme l'analyse quantitative non ciblée des métabolites d'un échantillon biologique, accède au cœur de nombreux processus physiologiques tels que la nutrition, l'adaptation biochimique et ultrastructurale ou la signalétique cellulaire. Les travaux réalisés constituent une approche de la physiologie du stress chez l'espèce considérée sensible /Arabidopsis thaliana/, par l'analyse du métabolome primaire dans une perspective exploratoire, diagnostique et corrélative
Abiotic stresses (extreme temperatures, salinity, drought) impair plant growth and development and are important agronomic challenges. Understanding stress responses developed by various species contributes to crop improvement and also constitutes an interesting approach of integrated mechanisms operating at different organisation levels in highly regulated ways. Availability of genotypes collections, molecular analysis tools and efficient bioinformatics pave the way to phenotyping of model plants at the genomic scale, in relation with their environment. Metabolmics, defined as unbiaised, nontargeted and exhaustive analysis of metabolites in biological samples, provide new insights into physiological processes like nutrition, biochemical and structural adaptation or cell signalling. Present work is an exploratory, diagnostic and correlative approach of primary metablome in the stress sensitive species Arabidopsis thaliana

Le 134Cs et le 137Cs, isotopes radioactifs du césium relâchés entre autres à la suite des accidents de Tchernobyl et Fukushima, sont des sources de préoccupations majeures pour la sécurité sanitaire et la protection des écosystèmes. La contamination des plantes est liée en partie à leur capacité à absorber le césium présent dans la solution du sol via des transporteurs. Le césium emprunte en effet, au moins en partie, le système de transport potassique sans disposer de transporteurs qui lui sont spécifiques. Il existe une grande diversité de transporteurs potassiques et la part du flux total qu’ils prennent en charge dépend du niveau de potassium fourni à la plante. Nous avons fait varier ce niveau et étudié son impact sur l’absorption et la distribution du césium dans la plante. Outre les phénomènes de compétition existant entre les deux éléments, le type de transporteurs dominant à un niveau de potassium donné a également une influence sur le transport du césium. Nous avons ainsi pu mettre en évidence des familles de transporteurs potassiques ayant de plus fortes chances d’être impliquées dans le transport de césium. Ainsi, les résultats produits pendant cette thèse mettent en évidence le rôle in planta du transporteur KUP9, jusque-là peu étudié, dans les flux de césium chez Arabidopsis thaliana. Nous n’observons pas de modifications de l’absorption du potassium chez les mutants invalidés sur ce transporteur : il serait donc possible de le manipuler pour moduler l’absorption de césium sans que la nutrition potassique ne soit altérée
134Cs and 137Cs, two radioactive isotopes unintentionally released after the Chernobyl and the Fukushima accidents, are of major concern for ecosystems protection and human health. Plants contamination is due to their ability to absorb cesium from the soil solution via transporters. Indeed, cesium which is supposed to have no role in plants can pass through potassium transporters. Proteins involved in potassium transport are diverse and the part of fluxes covered by each of them depends on the level of potassium supplied to the plant. We tested the effects of this level on uptake and distribution of cesium into the plant. Beside competition between the two elements, transporters which are dominant for a given potassium supply condition modify the cesium transport. Making the link between these modifications and knowledge on identity and properties of potassium transporters, we highlighted candidates with high potential for cesium transport. Hence, results produced during my thesis demonstrate in planta the role of KUP9 transporter, which has received little attention so far, in cesium fluxes in Arabidopsis thaliana. Changing in potassium uptake has not been observed in mutant lines disrupted in this KUP9 transporter suggesting interestingly that it could be possible to modulate cesium uptake without alteration of potassium nutrition

L'identification de genes intervenant dans l'acquisition du sodium (absorption, transport, stockage, excretion), a ete abordee par la recherche de mutants chez la plante modele arabidopsis thaliana (l. ), heynh. La selection des candidats mutants s'est faite sur la base de leur teneur foliaire en sodium tres superieure ou tres inferieure par rapport a celle du type sauvage, apres un traitement salin court (4 a 6 jours) et modere (concentration en chlorure de sodium 35 mm) en culture in vitro. Apres le criblage de 6625 plantes m2 issues d'une mutagenese a l'ems, 4 mutants suraccumulant le sodium dans leurs parties aeriennes ont ete isoles. L'etude genetique de l'un d'entre eux a montre que son phenotype mutant etait sous le controle d'un locus unique, recessif devant le type sauvage. Ce mutant a ete nomme sas1. Le locus sas1 a ete cartographie dans le bas du bras long du chromosome iii d'arabidopsis. L'accumulation foliaire de sodium chez sas1 est 2 a 10 fois superieure a celle des plantes sauvages, selon le systeme de culture utilise : culture in vitro, en hydroponie ou en serre sur terreau. Chez sas1, la suraccumulation foliaire concerne le sodium et son analogue, le lithium, mais pas le potassium, le magnesium ou le calcium. Par ailleurs, le mutant sas1 suraccumule le sodium dans tous ses organes aeriens (hypocotyle, feuille, tige, fleur, silique) mais non dans ses racines. Enfin, l'exsudat racinaire de sas1 a une concentration en sodium environ 5 fois plus elevee que celui des plantes sauvages. L'ensemble de ces donnees suggere que le mutant serait affecte dans une fonction specifique du sodium intervenant dans le chargement en sodium de la seve xylemique.