Universiẗat, Diss., 2004--Freiburg (Breisgau). / Erscheinungsjahr an der Haupttitelstelle: 2003.

Les plantes ont développé une forte capacité d'adaptation aux facteurs environnementaux comme les conditions nutritives. Les voies de signalisation qui perçoivent les signaux environnementaux et les intègrent au niveau du développement de la plante sont encore mal connues. La voie de signalisation TOR–S6Kinase qui est conservée au sein des eucaryotes, a été principalement étudié chez les animaux et la levure chez lesquels elle régule la croissance en réponse aux facteurs de l'environnement via le niveau de traduction, la synthèse des ribosomes et le cycle de division cellulaire. Chez l'angiosperme Arabidopsis thaliana, deux gènes codent pour des protéines S6Kinases mais les travaux publiés ne montrent pas une implication de ces deux gènes dans le développement de la plante. Notre travail a consisté à mettre en évidence l'implication des protéines S6Kinases chez les plantes en utilisant comme modèle la mousse Physcomitrella patens. Nous avons développé des conditions expérimentales pour étudier le développement du protonéma de mousse qui est constitué de deux types cellulaires, le chloronéma et le caulonéma. Par exemple, nous avons caractérisé un marqueur moléculaire du caulonéma, un type cellulaire induit en condition de carence. Nous avons identifié 3 gènes codants pour les protéines S6Kinases chez Physcomitrella patens puis, nous avons réalisé les trois simples mutants par transgénèse ciblée. Nos résultats indiquent que le gène PpS6K1 permet de réguler le développement du protonéma en fonction des conditions environnementales en jouant principalement sur le rythme de division des chloronémas en fonction des nutriments. / Plants have developed a strong capacity to adapt to environmental cues like nutritive conditions. However, the signalling pathways involved in the perception of environmental signals and their integration into plant development are still poorly understood. The TOR-S6kinase signalling pathway is conserved in all eukaryotes but has been mainly studied in yeast and animals where it is known to regulate growth in response to the environment via translation, ribosome synthesis and the cell cycle. In the angiosperm Arabidopsis thaliana, two genes encode S6 kinases but their functions during development are not known.The objective of this work was to characterise the function of S6 kinases in plants using the moss Physcomitrella patens as a model system. We have developed new methods to study the development of moss protonema, a filamentous tissue made of only two cell types: chloronema and caulonema. For example, we have characterized a molecular marker of caulonema, the cell type induced by starvation. We have characterized the three genes encoding P. patens S6 kinases and used gene targeting to generate knock-out mutants for each of them. Our results indicate that PpS6K1 regulates protonema development in response to nutrient conditions, mainly through the rate of chloronema cells proliferation. In the other hand, PpS6K2 is involved in the inhibition of the chloronema to caulonema transition and in nutrient sensing. PpS6K3 seems to be involved in the development of the gametophore and the sporophyte. Thus, our results show that the three S6Ks are involved at different levels in the regulation of growth and development in the moss P patens.

Universiẗat, Diss., 2002--Freiburg (Breisgau). / Erscheinungsjahr an der Haupttitelstelle: 2002.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Starch is a polyglucan made up of the two glucose polymers, amylose and amylopectin. Plants use starch to store excess carbohydrates from photosynthesis which get used for growth during the night. Starch metabolism is well undertood in higher plants such as A. thaliana thaliana and Solanum tuberosum with well-established pathways worked out for the enzymes involved in its synthesis and degradation. The bryophyte Physcomitrella patens has emerged as a popular choice for studying gene function in lower plants both because its genome has been sequenced and because of the ease of establishing knockout mutants via homologous recombination. Many metabolic functions have been studied in P. patens but, until now, little has been done in examining starch metabolism in moss. This study focused on two enzymes that have been found to be involved in starch degradation in higher plants, Disproportionating enzyme 1 (DPE1) and Disproportionating enzyme 2 (DPE2). DPE1 isoforms have been found to break down malto-oligosaccharides, which are products of starch degradation, into glucose within the chloroplast. On the other hand DPE2 catabolizes maltose to glucose in the cytosol. Higher plants that were silenced in these two genes were unable to degrade starch effectively, which lead to an increase in starch, malto-oligosaccharides or maltose and reduced growth. Three orthologs were identified for DPE1 in P. patens (PpDPE1A, B and C) and one for DPE2 (PpDPE2). Only PpDPE1B and PpDPE1C were found to be expressed in P. patens at the beginning of the light period but further investigation would be necessary at different time points as these genes were shown to be optimally expressed at the end of the light period. Targeted gene knockouts were made for each in P. patens which showed a reduced growth phenotype for all, indicating that these genes do play a role in starch catabolism that influences growth. There was, however, no significant change in starch content between the mutant lines and wild type (Wt). GFP fusion proteins showed PpDPE2 to be localized in cytosol, in close proximity to the chloroplast membrane. Similar findings have been found for DPE2 in A. thaliana and S. tuberosum. We hypothesize that PpDPE2 may play a role in cold tolerance in moss as an increase in starch breakdown has been witnessed in cold treated moss as well as increased transcript levels of starch metabolism genes and a maltose transporter. This opens a door to the further study of these generated mutant lines under cold stress. / AFRIKAANSE OPSOMMING: Stysel is ‘n poliglukaan wat bestaan uit die twee glukose polimere: amilose en amilopektien. Plante gebruik stysel om oortollige koolhidrate van fotosintese wat vir groei gebruik word gedurende die nag te berg. Styselmetabolisme in hoër plante soos A. thaliana thaliana en Solanum tuberosum word goed verstaan, met gevestigde paaie uitgewerk vir die ensieme wat betrokke is by die sintese en afbreek daarvan. Die briofiet Physcomitrella patens is ‘n populêre keuse vir die bestudering van geenfunksie in laer plante, omdat die genoomvolgorde bepaal is en as gevolg van die gemak waarmee ‘uitklop’-mutante via homoloë rekombinasie gevorm kan word. Baie metaboliese funksies is bestudeer in P. patens maar tot nou is min gedoen om die styselmetabolisme in mos te ondersoek. Hierdie studie het gefokus op twee ensieme, DPE1 and DPE2, wat gevind is om betrokke is afbreek van stysel in hoër plante. Dit is voorheen bevind dat DPE1 isoforme malto-oligosakkariedes (wat produkte is van styselafbraak) afbreek na glukose in the chloroplast. Aan die ander kant kataboliseer DPE2 maltose na glukose in die sitosol. Hoër plante waarin hierdie gene stilgemaak is, is nie instaat daartoe om stysel effektief af te breek nie. Dit lei tot ‘n verhoging in stysel, malto-oligosakkariede of maltose en verminderde groei. Drie ortoloë is geïdentifiseer vir DPE1 in P. patens (PpDPE1A, B en C) en een vir DPE2 (PpDPE2). Slegs PpDPE1B en PpDPE1C word uitgedruk in P. patens aan die begin van die ligperiode, maar verder ondersoek sal nodig wees op verskillende tydpunte, omdat dit bewys is dat hierdie gene optimaal uitgedruk word tydens die einde van die ligperiode. Geteikende geen uiklop-mutante is gemaak vir elk in P. patens wat ‘n verminderde-groei fenotipe vertoon het vir almal, wat aandui dat hierdie gene ‘n rol speel in styselkatobolisme wat groei beïnvloed. Daar was egter geen beduidende verskil in styselinhoud van die mutante lyne en die wilde tipe nie. GFP-fusieproteïne het gewys dat PpDPE2 gelokaliseer is in die sitosol, naby aan die chloroplast membraan. Soorgelyke bevindinge is ook gemaak in DPE2 in A. thaliana en S. tuberosum. Dit word gestel dat PpDPE2 moontlik ‘n rol speel in kouetoleransie in moss, omdat ‘n verhoging in styselafbraak opgemerk is in koue-behandelde moss sowel as verhoogde transkripsievlakke van styselmetabolisme gene en ‘n maltose transporter. Dit maak ‘n deur oop vir verdere studie van hierdie gegenereerde mutant-lyne onder kouestres.

The discovery of a mammalian endocannabinoid, anandamide (AEA or NAE 20:4) in Physcomitrella patens but not in higher plants prompted our interest in characterizing its metabolism and physiological role in the early land plants. Anandamide acts as an endocannabinoid ligand in the mammalian central and peripheral systems and mediates various physiological responses. Endocannabinoid signaling is terminated by a membrane-bound fatty acid amide hydrolase (FAAH). Using in silico analyses, we identified nine orthologs of human and Arabidopsis FAAH in P. patens (PpFAAH1 to PpFAAH9). Predicted structural analysis revealed that all the nine PpFAAH contain characteristic amidase signature sequence with a highly conserved catalytic triad and share a number of key features of both plant and animal FAAH. These include a membrane binding cap, membrane access channel, substrate binding pocket and as well as potential for dimerization. Among the nine, gene expression for PpFAAH1 and PpFAAH9 was enhanced with exogenous AEA treatment. Cloning and heterologous expression, followed by radiolabeled in vitro enzyme assays revealed that PpFAAH1 activity was optimal at 37 °C and pH 8.0. Furthermore, PpFAAH1 showed higher specificity to NAE 20:4 than to other N- acylethanolamines such as NAE 16:0. Highest in planta amide hydrolase activity was noted in microsomes of gametophytes, suggesting the possibility for membrane localization of active FAAH. Interestingly, when FAAH1 was overexpressed, the moss cultures not only showed reduced growth but their transition from protonema to gametophyte was inhibited, which was rescued by exogenous AEA. Unlike overexpressors of AtFAAH1, which showed enhanced growth and hypersensitivity to abscisic acid, PpFAAH1 overexpressors showed tolerance to abscisic acid. Together, these data suggest that the occurrence of anandamide and distinct properties of PpFAAH1 in early land plants have physiological implications that are different from that of higher plants.

No description available.

No description available.

Cell division is an important biological process, thus it is always an active field in biological research. To complete cell division, plant cells form a new cell wall that separates the two new cells. In contrast to the contractile ring of animal cells, plant cells form the new cell wall from their interior. Vesicles containing the new cell wall fuse at the cell plate between the two cells. The formation of the cell plate is guided by the phragmoplast, a microtubule and filamentous actin-containing structure. Because vesicles are known to be transported by myosin motors during interphase and little is known about the role of myosin XI during cell division, I investigated the participation of the plant specific myosin XI in cell division. For this work I used the moss Physcomitrella patens as a model organisms because of its simple cytology and powerful genetics. Using a fluorescent protein fusion of myosin XI, I found that this molecule associates with the mitotic spindle immediately after nuclear envelope breakdown. Myosin XI stays associated with the spindle during mitosis, and when the phragmoplast is formed, it concentrates at the cell plate, forming a fine line. Using an actin polymerization inhibitor, latrunculin B, I found that the associations of myosin XI with the mitotic spindle and the phragmoplast are independent of the presence of filamentous actin. After using double-labeled lines for myosin XI the endoplasmic reticulum and vesicle markers, I found the myosin XI on the spindle is not colocalized with the endoplasmic reticulum and two types of vesicle markers. Furthermore, I also found the vesicle trafficking inhibitor, brefeldin A, does not inhibit the localization of myosin XI at the mitotic spindle and the phragmoplast. These observations suggest a new actin-independent behavior of myosin XI during cell division, and provide novel insights to our understanding of the function of myosin XI during plant cell division.

No description available.

No description available.