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1
Mittmann, Franz. « Molekularbiologische Untersuchungen zum Phytochromsystem der Moose Physcomitrella patens und Ceratodon purpureus ». [S.l.] : [s.n.], 2002. http://www.diss.fu-berlin.de/2003/94/index.html.
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Cast, Delphine. « Régulation de la croissance : Implication des protéines ribosomales S6Kinases chez la mousse Physcomitrella patens ». Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4095.
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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 nutrimentsPlants 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
3
Russell, Angela Julia. « Morphogenesis in the moss Physcomitrella patens ». Thesis, University of Leeds, 1993. http://etheses.whiterose.ac.uk/1535/.
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A method was developed for recording the development of moss protonema using time-lapse video microscopy. This has provided a detailed record of the time-course of development from spore germination to the production of gametophores. Detailed records of the growth of primary and secondary chloronema, the transition of primary chloronema to caulonema, and the development of side-branches were obtained. Filaments were found to undergo the transition to caulonema earlier than previously thought. The majority of caulonemas ide-branches were found to begin as chloronema and switch to caulonema after one or two cell cycles. The early cell divisions of bud formation were found to follow a distinct pattern, which was upset by high concentrations of cytokinin and lanthanum. The response of caulonema apical cells to polarotropic light was recorded and compared to the gravitropic response. The time-lapse studies provided the basis for the further development of the quantitative analysis of protonemal branching patterns to include second and third side-branches of a sub-apical cell, and transitional caulonema. Analysing side-branch patterns should allow the detection of developmental mechanisms underlying the determination of side-branch fate. The potential of this method for assessing the effect of hormone treatments and for analysing more precisely mutant phenotypes was explored. An analysis of bud spacing was carried out to determine if the formation of a bud on a filament was inhibitory to other buds forming on the same filament. It was found, to the contrary, that buds tended to form in clusters. The hypothesis that the primary mode of action of cytokinin is an enhanced influx of calcium ions into the cell was investigated. Classical electrophysiology was used in order to detect any change in membrane potential suggestive of ionic fluxes in response to cytokinin treatment. No definitive changes in membrane potential were detected in response to cytokinin. This appeared to rule out the involvement of voltage-regulated channels in cytokinin action. The effects of some inhibitors used in studies of calcium on the moss protonemal system were examined. It is suggested that the concentrations commonly used had toxic effects that were not specific to calcium channels. The ionophore A23187 was used to characterise the protonemal response to a sustained influx of calcium. Some mutant strains were found to have a differential response to the ionophore. This may mean that they have mutations affecting their calcium regulatory system. Two new techniques of imaging calcium were used in order to detect changes in intracellular calcium in response to cytokinin. A method was developed for loading the dual wavelength fluorescent dye Indo-1 into moss protonema using iontophoretic microinjection, and intracellular calcium was imaged using ratio-image technology. Wild-type moss and some mutant strains were also successfully transformed with the gene for apoaequorin, and calcium luminescence measured in response to cold-shock and plant hormones. Some different responsesto temperatures hock were apparent in one of the transformed mutants. Preliminary experiments did not reveal any aequor independent calcium luminescence in response to cytokinin.
4
Knight, C. D. « Gravitropism in the moss Physcomitrella patens ». Thesis, University of Leeds, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383268.
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Lee, Kieran J. D. « The cell wall of Physcomitrella patens ». Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405745.
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Liénard, David. « Aquaporines et évaporation chez Physcomitrella patens ». Rouen, 2006. http://www.theses.fr/2006ROUES004.
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P. Patens est une plante poïkilohydre dont toutes les cellules sont en contact avec le milieu extérieur. Nous avons voulu déterminer l'influence des aquaporines au cours de l'évaporation des pseudo feuilles de ses gamétophores. Les aquaporines ont été recherchées dans une banque d'EST de P. Patens. Quatre aquaporines ont été clonées et nous avons obtenu des mutants knock-out pour trois d'entre elles (Pip2;1, Pip2;2 et Pip2;3). Les protoplastes des gamétophores des plantes mutantes correspondantes, pip21 et pip22, présentent une forte diminution de perméabilité, alors que la mutation demeure sans effet sur pip23. Ces plantes ne présentent pas de phénotype visuel lorsqu'elles demeurent dans une atmosphère saturée. Par contre lorsqu'elles sont soumises à un stress hydrique, pip21 et pip22 flétrissent plus facilement que le WT. Nous proposons un modèle pour expliquer cet effet. Nos mesures suggèrent également une activation réciproque entre pip21 et pip22Poikilohydric plants such as the moss P. Patens, which do not control their water loss, cannot regulate their water potential. We focused our work on the identification of aquaporins involved during evaporation from the pseudo gametophytic leaves of P. Patens. Four aquaporins Pip1;1, Pip2;1, Pip2;2 and Pip2;3, were cloned and knock-out mutations were obtained for three of them (Pip2;1, Pip2;2 et Pip2;3). Protoplasts from the corresponding mutant plants pip21 and pip22, exhibited a strong decrease in their water permeability, while the pip23 protoplast permeabilities remained unaffected. No difference was visible between the wild type and mutants, when plants were grown under a saturated atmosphere. On the opposite, pip21 and pip22 were less resistant than wild type to a water stress. We proposed a model to explain the role of these aquaporins during evaporation. Our measurements also suggest that interactions enhancing their permeabilities should exist between pip21 and pip22
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Faltusz, Alexander. « Molekulare und funktionelle Analyse von P-Typ-Kalzium-ATPasen im Laubmoos Physcomitrella patens (Hedw.) B.S.G ». [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971028567.
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Henschel, Katrin Andrea. « Strukturelle und funktionelle Charakterisierung von MADS-Box-Genen aus dem Laubmoos Physcomitrella patens (Hedw.) B.S.G ». [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=965796779.
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Wanke, Dierk. « Studien zur pflanzenspezifischen WRKY-Transkriptionsfaktorfamilie vergleichende Analyse zwischen dem Moos, Physcomitrella patens, und höheren Pflanzen sowie eine gesamtgenomische Betrachtung von WRKY-DNA-Bindungsstellen / ». [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=971303991.
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Ring, Andreas. « Serine/Arginine-rich proteins in Physcomitrella patens ». Thesis, Linköpings universitet, Molekylär genetik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-80870.
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Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in the flowering plant Arabidopsis thaliana. But so far no attempts on characterizing SR-proteins in the moss Physcomitrella patens have been done. SR-proteins are a conserved family of splicing regulators essential for constitutive- and alternative splicing. SR-proteins are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a form of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P. patens. The three genes show conserved intron-exon structure and protein domain distribution, not surprising since the gene family has evidently evolved through gene duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also been seen in A. thaliana. SR-proteins determine splice-site usage in a concentration dependent manner. SR-protein overexpression experiments in A. thaliana and Oryza sativa have shown alteration of splicing patterns of endogenous SR-proteins. Overexpression of PpSR40 did not alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that they might not be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may provide insights on the SR-protein regulation mechanisms of the common land plant ancestor.
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Kilaru, Aruna, Jedaidah Chilufya, S. Swati, Imdadul Haq, Suhas Shinde, L. Vidali et Ruth Welti. « Emerging Implications Of Anandamide In Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4791.
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Gömann, Jasmin [Verfasser]. « Sphingolipid biosynthesis in Physcomitrella patens / Jasmin Gömann ». Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1236401794/34.
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Hooper, Erica Jane. « Ecological genetics of the moss Physcomitrella patens ». Thesis, University of Leeds, 2008. http://etheses.whiterose.ac.uk/286/.
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Molecular genetic studies using the model bryophyte Physcomitrella patens have advanced the body of knowledge surrounding plant functional genetics and molecular biology, yet very little is known about the ecology and population genetics of this species. Although the bryophytes are the second largest group of plants, there is little information regarding the population genetics of bryophytes in general. To address these issues I have conducted the first study into the population genetics of P. patens, where plants from eight populations in Britain have been collected. Sampling within these populations was conducted according to a hierarchical scale, so as to assess not only the level of genetic variation within populations, but how this is structured spatially. Analysis of the plants collected was conducted using amplified fragment length polymorphism (AFLP) analysis. No spatial genetic structure was found within populations of P. patens, and it is hypothesised that the nature of the ephemeral aquatic habitats that P. patens occupies may account for this finding. In this thesis a novel method for studying the mating systems operating within bryophyte populations has been proposed, which exploits the dominant haploid stage of the bryophyte life cycle. This methodology has been applied to natural populations of P. patens, and evidence of mixed mating has been observed. Bryophytes are often overlooked or under-recorded in their natural environment, and distribution data within Great Britain is likely to be inaccurate for a large number of species. This issue is highlighted in this thesis, as a bryophyte species new to Europe has been discovered.
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Chilufya, Jedaidah Y. « Anandamide-Mediated Growth Changes in Physcomitrella patens ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etd/3162.
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Anandamide (NAE 20:4) or arachidonlyethanolamine (AEA) is the most widely studied N-acylethanolamine (NAE) because it mediates several physiological functions in mammals. In vascular plants, 12-18C NAEs inhibit growth in an abscisic acid (ABA)-dependent and -independent manner. Anandamide, which is unique to bryophyte Physcomitrella patens, inhibited gametophyte growth and reduced chlorophyll content when applied exogenously. It is hypothesized that anandamide mediates its responses through morphological and cellular changes. Following growth inhibition by short-term anandamide-treatment, microscopic analyses revealed relocated chloroplasts and depolymerized F-actin in protonemal tips. Long-term treatment showed partially bleached gametophyte cells with degraded and browning chloroplasts. These anandamide-mediated responses have physiological implications as AEA may function as a signal for gametophytes to activate secondary dormancy as seen with ABA. Future studies will investigate the role of AEA in mediating stress responses and possible interaction with ABA.
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Kilaru, Aruna. « Emerging Implications of Anandamide in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4770.
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Brücker, Gerhard. « Zell- und molekularbiologische Untersuchungen zum Photo- und Polarotropismus in den Moosen Ceratodon purpureus und Physcomitrella patens ». [S.l. : s.n.], 2003. http://www.diss.fu-berlin.de/2003/149/index.html.
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White, Christopher David. « The role of CLAVATA signalling in Physcomitrella patens ». Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708872.
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Mohensi, Kousha, et Aruna Kilaru. « Determination of Fatty Acid Composition in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/4845.
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Gautam, Deepshila, Imdadul Haq et Aruna Kilaru. « Phenotypic Characterization of FAAH Mutants in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/etsu-works/7733.
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Guatam, Deepshila, Imdadul Haq et Aruna Kilaru. « Phenotypic Characterization of FAAH Mutants in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/etsu-works/7734.
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Stander, Emily Amor. « Roles of disproportionating enzymes in the moss Physcomitrella patens ». Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/97992.
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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.
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Kilaru, Aruna, et Imdadul Haq. « Functional Characterization of Anandamide Hydrolyzing Enzyme in Physcomitrella patens ». Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/7728.
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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.
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Olsson, Tina. « Functional characterization of hexokinases in the moss Physcomitrella patens / ». Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2005. http://epsilon.slu.se/200578.pdf.
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Haq, Imdadul, Suhas Shinde et Aruna Kilaru. « Characterization of Fatty Acid Amide Hydrolase in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/4818.
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In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5‟- and 3‟-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with N-terminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.
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Chilufya, Jedaidah, et Aruna Kilaru. « Analyses of Anandamide-Mediated Growth Inhibition in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4830.
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In higher plants, a class of bioactive fatty acid ethanolamides or N-acylethanolamines (NAEs) mediate growth, development, cellular organization and response to stress, in an abscisic acid (ABA)-dependent or independent manner. Unlike in higher plants, Physcomitrella patens, a bryophyte contains anandamide or NAE 20:4, a mammalian endocannabinoid ligand that mediates a multitude of physiological functions including development and stress. Unique lipids in mosses are considered vital for their resilience to environmental stresses; such lipids might enable them to recognize stress at the cellular level, and respond with membrane reorganization and altered growth. Since the identification of anandamide in moss, we have shown that, like abscisic acid (ABA), it inhibits gametophyte growth in a dose-dependent manner and reduced chlorophyll content. It is hypothesized that moss gametophores undergo morphological and cellular changes during anandamide-mediated growth inhibition. To test this, gametophyte growth and morphological changes in phyllodes, under different concentrations of NAE 20:4, were digitally captured using Canon EOS 70D, and analyzed using ImageJ software. NAE 20:4 but not its free fatty acid, arachidonic acid, not only inhibited growth of both shoots and rhizoids in a dose-dependent manner but also showed remarkable cellular changes. Phyllodes and protonemal cells of NAE 20:4 treated plants were further examined under stereo and compound light microscopes. Long- and short-term treatment with anandamide resulted in reduced chloroplast number, cytoplasmic shrinkage and plasmolysis in phyllodes and protonemal cells. A 100 micromolar NAE 20:4 treatment resulted in complete loss of green pigmentation in phyllodes. Effects of anandamide on cytoskeletal organization will be studied using Physcomitrella plants expressing GFP-talin and tubulin, via confocal microscopy. Together, these data will provide insights into anandamide-mediated cellular responses during growth inhibition.
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Gütle, Desirée. « Characterization of the ferredoxin/thioredoxin system and its targets in Physcomitrella patens ». Thesis, Université de Lorraine, 2017. http://www.theses.fr/2017LORR0055/document.
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La régulation redox est un mécanisme ancien présent chez les organismes biologiques et impliquée dans diverses voies métaboliques. En particulier chez les organismes photosynthétiques elle est responsable des mécanismes d‘adaptation rapide dans un environnement constamment modifié. Dans les chloroplastes le système ferrédoxine/thiorédoxine est la cascade redox principale qui relie l‘activité de plusieurs enzymes plastidiales à la source lumineuse. Le rôle central dans ce système est joué par la ferrédoxine-thiorédoxine réductase (FTR), une protéine hétérodimérique qui récupère des électrons à partir de la ferrédoxine photoréduite et les transfère pour réduire des thiorédoxines plastidiales. Ces protéines peuvent alors réduire des enzymes cibles, requérant l‘accessibilité de paires de cystéines dans un disulfure dont la réduction résulte en une activation/ inactivation de la cible. Jusqu‘à présent des plantes viables n‘ont pu être obtenues en l‘absence de ce système de régulation. Dans cette thèse des secteurs du système redox ont été explorés chez la plante modèle Physcomitrella patens (une mousse). Par manipulation de gènes l‘influence de l‘enzyme FTR sur la croissance et le développement de la plante a été analysée suivant différents paramètres. De manière à impacter la fonction de la réductase des changements nucléotidiques simples ont été introduits au niveau des codons programmant les cystéines catalytiques et dans un deuxième temps le gène complet a été supprimé. De façon inattendue nous n‘avons observé aucun effet significatif sur la viabilité et le développement des plantes mutantes. De plus, nous avons détecté dans P. patens des thiorédoxines additionnelles absentes chez les plantes à graine qui sont fonctionnelles vis à vis des enzymes cibles mais non-réduites par la FTR. Ceci rend possible un scénario de compensation chez les mutants via un système de réduction FTR-indépendant qui reste à caractériser. Deux des cibles photorégulées, la fructose-1,6-bisphosphatase (FBPase) et la sédoheptulose-1,7-bisphosphatase (SBPase), fonctionnent dans la phase de régénération du cycle de Calvin-Benson cycle et elles possèdent plusieurs caractéristiques de catalyse et de régulation similaires. En combinant des approches biochimiques et structurales, une comparaison fonctionnelle et structurale des deux phosphatases de P. patens a été conduite. De plus l‘analyse phylogénétique a révélé une origine procaryotique indépendante des deux séquences en dépit de leurs similitudes structurales et catalytiques. De plus trois articles de revue résument la plasticité et la représentativité du modèle P. patens pour la recherche forestière, les principes généraux de la régulation redox relativement aux aspects évolutifs et fonctionnels chez les plantes ainsi que l‘ état de l‘art de la régulation redox chez les espèces ligneuses en utilisant principalement le peuplier comme modèleRedox regulation is an ancient mechanism present in biological organisms and is involved in diverse cellular pathways. In particular in photosynthetic organisms it is responsible for fast adaption mechanisms to a constantly changing environment. In chloroplasts the ferredoxin/thioredoxin system represents the main redox regulatory cascade which links the activity of several plastid enzymes to the energy source, light. A central role in this system is played by the heterodimeric ferredoxin-thioredoxin reductase (FTR), which gains electrons from the photo-reduced ferredoxin and transfers those further on via reduction to plastidal thioredoxins. Those proteins in turn reduce their target enzymes and require therefore the availability of redox sensitive cysteine pairs whose reduction results in an inactivation/activation switch of the targets. So far no viable plants could be obtained in complete absence of this redox regulation system. In this thesis single sections of the system were explored in the model plant Physcomitrella patens. Through gene manipulation the influence of the FTR enzyme on plant growth and development was analysed. In order to impact on the function of the reductase, firstly single nucleotide exchange of the catalytic cysteines was performed and later on the gene was completely deleted. Surprisingly, no significant effect could be observed on the viability and development of mutant lines compared to WT plants. Furthermore we found that P. patens possesses in contrast to seed plants additional thioredoxins which are functional for reduction of FTR target enzymes but are most likely not supplied with electrons by this reductase. Thus a possible rescue scenario independent of FTR could be assumed for P. patens and also by other redox regulation systems present in chloroplasts. Two of the FTR target enzymes, fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase, are functional in the regeneration phase of the Calvin-Benson cycle and share similar characteristics in regulation and catalysis. By combining biochemical and structural approaches, a functional comparison of both phosphatases was conducted using cDNAs from P. patens. A stricter TRX-dependent regulation and catalytic cleavage ability for both substrates, FBP and SBP, could be observed for PpSBPase, whereas PpFBPase is only capable of cleaving FBP. By obtaining the oxidized X-ray structure of both enzymes these observations can be associated with the distinct positions of regulatory sites and the various sizes of the substrate binding pocket. In addition, the phylogenetic analysis revealed an independent prokaryotic origin for both phosphatases. Furthermore we summarized in three review articles the amenability of P. patens as model plant for forest research, the general principles of redox regulation in respect of evolution and functional mechanisms in plants, and the current state of the art in forest redox regulation using poplar as exemplary model
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Haq, Imdadul, et Aruna Kilaru. « Fatty Acid Amide Hydrolase in an Early Land Plant, Physcomitrella patens ». Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/7727.
Texte intégralRésumé :
Fatty acid amide hydrolase (FAAH) belongs to a diverse class of enzymes in amidase signature family. In mammals, FAAH is targeted to affect neurological functions because it terminates endocannabinoid signaling by degrading anandamide, a 20C N-acylethanolamine (NAE 20:4). In higher plants, FAAH is known to modulate growth, development and stress tolerance by degrading 12-18C NAEs. Since anandamide was reported to exclusively occur in early land plants, we investigated its metabolic pathway in the moss Physcomitrella patens. Based on the highest identity with ratFAAH, we identified nine orthologs in moss, PpFAAH1 to PpFAAH9. Several bioinformatic tools were used to understand the structural basis of how catalytic residues fold for amidohydrolase activity. Based on these in silico analyses of PpFAAH homologs and their gene expression in response to saturated (NAE16:0) and unsaturated NAE (NAE 20:4) treatment, PpFAAH1 was selected for biochemical characterization. Heterologously expressed PpFAAH1 showed highest amidohydrolase activity at 37°C and pH 8.0. Both in vivo and in vitro studies showed that unsaturated NAE substrate is hydrolyzed faster than the saturated NAE (> 10-fold in vivo and 50-fold in vitro). Additionally, transgenic moss lines over expressing FAAH1 showed slower growth and disrupted gametophyte formation when compared to wild type. These data suggest that PpFAAH1-mediated NAE metabolism is likely involved in developmental transition in moss.
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Rödel, Philipp [Verfasser], et Ralf [Akademischer Betreuer] Reski. « Quantitative Analyse des auxininduzierten miR160/ARF-Regelkreises in Physcomitrella patens ». Freiburg : Universität, 2012. http://d-nb.info/1123470960/34.
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McClelland, D. J. « Genetical studies of gametophyte development in the moss Physcomitrella patens ». Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233202.
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Shinde, Suhas, Jedaidah Chilufya, Shivakumar Devaiah, Ruth Welti et Aruna Kilaru. « Anandamide-Mediated Growth, Morphological and Cellular Changes in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/4785.
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Chilufya, Jedaidah, S. Khurana, L. Vidali et Aruna Kilaru. « Anandamide-Mediated Growth, Morphological And Cellular Changes In Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4792.
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Swati, Swati, et Aruna Kilaru. « Biochemical Characterization of Fatty Acid Amide Hydrolase in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4829.
Texte intégralRésumé :
N-acylethanolamines (NAEs) are fatty acid ethanolamides that mediate stress responses in plants and animals. NAEs such as NAE 20:4 (anandamide) have only been reported in mammals and they regulate processes like neuroprotection and pain perception. Interestingly, we discovered the unique occurrence of anandamide in moss, Physcomitrella patens, a stress tolerant early land plant. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. I specifically propose to biochemically characterize FAAH enzyme that degrades anandamide. For this, Arabidopsis FAAH (AtFAAH) homolog was identified in moss database using BLASTP. The predicted protein structure of putative moss FAAH (PpFAAH) closely resembled to that of AtFAAH with conserved amidase signature sequence and catalytic triad residues: Lys205, Ser281, Ser305. Transcript levels of PpFAAH increased five-fold when moss was grown on excess NAE containing media. PpFAAH cDNA was PCR amplified and cloned into pET23a expression vector and transformed into RIL E. coli cells and confirmed by colony PCR. Heterologously expressed protein will be purified by Ni+2 affinity column chromatography and confirmed by western blot using anti-His-tag antibody. For biochemical characterization, enzyme will be presented with 14C NAE 20:4 substrate and rate of product free fatty acid formed will be quantified by extracting lipids from reaction mixture and separating by thin layer chromatography followed by radiometric scanning. E. coli cells expressing AtFAAH enzyme will be used as control. A complete characterization of the PpFAAH enzyme will be carried out to determine the kinetics, optimal temperature and pH conditions. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.
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Swati, Swati, et Aruna Kilaru. « Biochemical Characterization of Fatty Acid Amide Hydrolase in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/4841.
Texte intégralRésumé :
N-acylethanolamines (NAEs) are a group of fatty acid ethanolamides and their metabolic pathway is highly conserved in eukaryotes. However, metabolites such as NAE 20:4 (anandamide) are known to occur in mammalian systems but not in higher plants. Anandamide is an endocannabinoid receptor ligand and mediates stress responses and regulates various physiological processes such as neuroprotection, pain perception and appetite suppression in animals. Interestingly anandamide occurrence was recently reported in a highly stress tolerant early land plant, Physcomitrella patens but its physiological role remains to be elucidated. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. To test this hypothesis, arabidopsis FAAH homolog was used to search moss database using BLASTP. Eight putative FAAH candidates (PpFAAH1-8), with an amidase signature sequence and conserved catalytic sites, were identified. Among these, PpFAAH1 and PpFAAH2 responded to exogenous NAE, and their 3D predicted protein structure closely resembled to that of AtFAAH1. The 1.8Kb coding region of putative PpFAAH1 was chosen for further characterization and was PCR amplified, cloned into TrcHis2 expression vector and transformed into E. coli TOP10 cells. Upon confirmation of the positive clones and induction of proteins, expressed proteins will be purified by Ni+2 affinity column chromatography, confirmed by western blot and analyzed for its substrate specificity using radiolabelled anandamide. Lipids extracted from reaction mixture will be separated by thin layer chromatography and detected by radiometric scanning. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.
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Shinde, S., R. Welti et Aruna Kilaru. « Novel Polyunsaturated N-acylethanolamines and Their Implications in Physcomitrella patens ». Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etsu-works/4867.
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Kilaru, Aruna, et Imdadul Haq. « Characterization of a Mammalian Endocannabinoid Hydrolyzing Enzyme in Physcomitrella patens ». Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/7722.
Texte intégralRésumé :
The discovery of a mammalian endocannabinoid, anandamide (N-arachidonylethanolamide; 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 plant. 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). Based on sequence identity and 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 levels for PpFAAH1 and PpFAAH9 were enhanced with exogenous anandamide treatment. Further 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 gametophyte tissues, 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 protonemal stage to gametophyte was inhibited, which was rescued in part 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.
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Swati, Swati. « Cloning of N-acylethanolamine Metabolic Pathway Genes from Physcomitrella patens ». Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etd/3178.
Texte intégralRésumé :
N-acylethanolamines (NAEs) including anandamide are lipid derivative molecules, which play vital roles in physiological and developmental processes in plants and animals and mediate stress responses. In mammals, NAEs are synthesized from hydrolysis of their precursor molecule N-acylphosphatidylethanolamine (NAPE) by NAPE-specific phospholipaseD (NAPE-PLD). All NAEs including anandamide (NAE20:4) are hydrolyzed by fatty acid amide hydrolase (FAAH) into free fatty acid and ethanolamine. To date, different NAEs including anandamide have been identified in Physcomitrella patens but its metabolic pathway remains undiscovered. It is hypothesized that NAE metabolic pathway in P. patens is conserved and is similar to that of other eukaryotic systems. To this extent, putative PpNAPE-PLD and PpFAAH were identified and cloned for heterologous expression and characterization. Expression of PpFAAH was further verified by Western blot analysis. Future studies will involve biochemical characterization of putative PpNAPE-PLD and PpFAAH, to establish the evolutionarily conserved nature of NAE functions in early land plants.
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Shinde, Suhas, Ruth Welti et Aruna Kilaru. « NOVEL POLYUNSATURATED N-ACYLETHANOLAMINES AND THEIR IMPLICATIONS IN PHYSCOMITRELLA PATENS ». Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/asrf/2018/schedule/111.
Texte intégralRésumé :
N-Acylethanolamines (NAEs), although are ubiquitous in plants and animals the occurrence of endocannabinoid ligands and the corresponding cannabinoid receptors was limited to mammals. Interestingly, bryophytes, unlike seed plants possess arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5), which are fatty acid precursors for endocannabinoid ligands. Here, we show that the moss Physcomitrella patens contains ~24 and 7 % of AA and EPA, respectively. Using selective lipidomic profiling, we identified polyunsaturated NAEs, including N-arachidonoyl ethanolamide (anandamide/AEA/NAE 20:4) and N-eicosapentaenoyl ethanolamide (EPEA) and also their corresponding N-acyl-phosphatidylethanolamine (NAPE) precursors in various developmental stages of Physcomitrella. Quantification of various NAPE and NAE species indicated the abundance of unsaturated species over saturated. In all haploid developmental stages analyzed, NAE 20:4 levels contributed to ~ 30 % (~ 26 ng mg-1 lipid) of the total NAE while NAE 20:5 remained as a minor component (~ 5 %; ~ 4.5 ng mg-1 lipid). Exogenous application of AEA, EPEA and their corresponding fatty acid precursors (AA and EPA, respectively) inhibited the growth of gametophytes and protonemata in a dose-dependent manner. AEA has shown the exclusive effect on the F-actin dynamics at the apex of protonemal cells, which was similar to the effect of abscisic acid (ABA) on protonemal growth inhibition. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that was responsive to exogenous ABA. Putative PpNAPE-PLD was expressed in E. coli for further characterization. Our data demonstrate the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with the occurrence of AEA and EPEA.
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Sun, Hao. « Analysis of Myosin XI Localization During Cell Division in Physcomitrella patens ». Digital WPI, 2015. https://digitalcommons.wpi.edu/etd-theses/783.
Texte intégralRésumé :
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.
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Zobell, Oliver. « The family of CONSTANS like genes in the moss Physcomitrella patens ». [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=979484707.
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Yasumura, Yuki. « Conserved regulation of chloroplast development in 'Physcomitrella patens' and higher plants ». Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404280.
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Boyd, P. J. « Changes in gene expression during development in the moss, Physcomitrella patens ». Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233170.
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Haq, Imdadul, Suhas Shinde et Aruna Kilaru. « Fatty Acid Amide Hydrolase In Nae Metabolic Pathway In Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/4786.
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Haq, Imdadul, Suhas Shinde et Aruna Kilaru. « Fatty Acid Amide Hydrolase in Nae Metabolic Pathway in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etsu-works/4815.
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Farley, C., Aruna Kilaru, Shivakumar Devaiah, M. Roth, A. Shiva, P. Tamura et Ruth Welti. « Composition of N-Acylethanolamines in Physcomitrella Patens at Varying Life Stages ». Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etsu-works/4825.
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Sante, Richard, et Aruna Kilaru. « The Role of N-Acylethanolamines in the Development of Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/4864.
Texte intégralRésumé :
Global demand for food, which is expected to double by 2050, presents plant biologists with a major challenge to generate higher yields on existing croplands. The long-term goal of the proposed research is to generate stress tolerant plants and improve crop productivity. Stress responses, in plants or animals, most often involve the activation stress-signaling pathways. One pathway is mediated by a group of bioactive lipid molecules, N-acylethanolamines (NAEs), and is highly conserved among eukaryotes. NAEs are composed mainly of amides of ethanolamine and fatty acids. In model Arabidopsis thaliana (At), NAEs inhibit germination and growth in seedlings via abscisic-dependent and independent mechanisms aimed at stress regulation. This work focuses on identifying the NAE pathway in the moss, Physcomitrella patens (P. patens), that shows higher resistant to stress than At and consequently the role played by NAE metabolites in the development of P. patens. Mosses are small seedless and fruitless early plants that have evolved successful mechanisms of surviving prolonged stresses during their transition from aquatic life to land. Previous studies have shown that P. patens is tolerant to high temperature, salinity and osmotic shock, to which most plants are fragile. I hypothesize that the NAE pathway is present in P. patens and that it plays a significant role in its development. NAE composition and metabolizing enzymes like FAAH and NAPE synthase will be measured to ascertain NAE pathway in P. patens. Further quantification of NAE content and composition during development will identify the key developmental time points that are associated with high metabolite levels. Total lipids will be extracted in triplicates from twelve tissue samples harvested at protonema stage; mature gametophyte stage which is subdivided into leafy tissue and rhizoids; antheridia and archegonia stage; zygotic stage and the mature and germinating developmental stages. The high lipid content of the mature spores was previously associated with extreme longevity in ephemeral habitats. It would be of interest to determine if such an association exists with NAE metabolite content. Key time points will then be used to determine the effect of exogenous NAE 12:0. Preliminary analysis shows that P. patens have higher levels of NAEs than other plants examined. Furthermore, fatty acid amide hydrolases (FAAH) that hydrolyses NAEs were identified in silico. Putative PpFAAH1 candidate showed 95 % homology with previously characterized AtFAAH in At. These results show the occurrence of NAEs in P. patens and a possible enzyme for its hydrolysis. Further confirmation of the NAE pathway in P. patens will be done by in silico identification of the precursor enzyme NAPE synthase. Understanding how P. patens tolerates stress using NAEs pathways by identifying key time points in its development will help other researchers know which specific NAEs affect the growth of P. patens and will facilitate the characterization of metabolic enzymes at specific times.
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Wessel, Tim. « Charakterisierung der beta-Untereinheit von heterotrimeren G-Proteinen aus Physcomitrella patens ». [S.l. : s.n.], 2003. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10316291.
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Stevenson, Sean Ross. « Forward genetics analysis in Physcomitrella patens identifies a novel ABA regulator ». Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/11876/.
Texte intégralRésumé :
Land plants evolved from a group of aquatic algae known as charophytes and molecular evidence suggests that they were pre-adaptated to life on land. Early land plants necessarily required mechanisms to survive dehydration and the plant hormone abscisic acid (ABA) is known to play a vital role in this conferring desiccation tolerance in all land plants. The basal non-vascular land plants, made up of the liverworts, hornworts and mosses, rely heavily on ABA-mediated vegetative dehydration/desiccation tolerance (D/DT) as they lack anatomical adaptations to retain water and this trait remains a developmentally regulated feature of the angiosperm seed. ABA non-responsive (anr) mutants were identified in the model bryophyte Physcomitrella patens and genotyping of segregating populations enabled the mapping of the PpANR locus. This locus encodes a trimodular MAP3 kinase comprising an N-terminal PAS domain, a central EDR domain and a C-terminal MAPKKK-like domain (“PEK” structure). Mutants of PpANR showed dehydration hypersensitivity and an inability to respond to exogenous ABA demonstrating the vital role of PpANR in the ABA-dependent osmotic stress responses. RNA-seq analysis of wild-type and anr mutant plants also revealed potential components of the wild-type ABA-dependent osmotic stress response not yet characterised in bryophytes. Phylogenetic analysis reveals PpANR to be part of a basal plant-specific subfamily of MAP3Ks closely related and possibly ancestral to the “EK” structured negative ethylene regulator CTR1 and the “PK” structured positive ABA regulators Raf10/11. The establishment of these subfamilies in the charophytes suggests them as potential vital components of ancestral water stress responses. The PAS domain likely originated from a domain swap from histidine kinases in the green algae and the solving of the crystal structure of this domain reveals it to form a homodimer with each domain taking the canonical PAS fold structure. A model is suggested for a key role of PpANR in an ancestral ABA-dependent osmotic stress signalling pathway.
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Haq, Md I., et Aruna Kilaru. « FATTY ACID AMIDE HYDROLASE IN NAE METABOLIC PATHWAY IN PHYSCOMITRELLA PATENS ». Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/asrf/2018/schedule/199.
Texte intégralRésumé :
In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 20C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, their metabolism and functional implications in plants however, remain incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. FAAH orthologs, putative PpFAAHs (Physcomitrella patens FAAH) were identified based on the sequence blast of ratFAAH, and named as PpFAAH1 to PpFAAH10. Based on the highest mRNA expression of the PpFAAH homologs upon NAE treatment, PpFAAH1 was selected for further in vitro characterization, which shares 31% sequence identity with ratFAAH. PpFAAH1 was heterologously expressed in E. coli and purified for characterization. Highest amidohydrolysis activity of PpFAAH1 was observed in vitro at pH 8.0 and temperature 37°C. Methoxy arachidonyl fluorophosphonate (MAFP), an inhibitor showed highest inhibition with 10mM concentration, however, one of the principal classes of FAAH inhibitor O-aryl carbamates (URB597) exhibited only 22% inhibition with the same concentration. Both in vivo and in vitro studies showed that unsaturated NAE substrate (NAE 20:4) is hydrolyzed faster than the saturated NAE (NAE16:0); more than 50- and 10-fold higher in vitro and in vivo assays, respectively. Amidohydrolase activity in vivo was mostly associated with microsomes compared with cytoplasmic fractions. Additionally, microsomal fraction of mature gametophytes showed higher amidohydrolase activity than of the protonemal or early gametophyte stages; however, PpFAAH expression was not significantly different between the developmental stages. Further functional characterization of NAE metabolic pathway is ongoing by generation of PpFAAH knock out (KO) and overexpressor (OE) to understand the biological implications of FAAH in growth and development of early land plants.
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Kilaru, Aruna, Imadadul Haq, Jedaidah Chilufya, Shivakumar Devaiah, Sushas Shinde et Ruth Welti. « Novel Polyunsaturated N-acylethanolamines (NAE) and Their Role in Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/4763.
Texte intégralRésumé :
Anandamide (N-arachidonoylethanolamide, AEA), a 20C polyunsaturated (PU) N-acylethanolamine (NAE) influences many neurological functions in mammals. Although 20C PU-NAEs are considered unique to animals, they were recently discovered in early land plants but their metabolism and functions remain unknown. Comprehensive lipidomic analyses of Physcomitrella patens revealed not only abundance of arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5) but also their corresponding ethanolamides (AEA and EPEA, respectively). While moss showed increasing AA with development, 14% and 24% in protonemata and gametophyte tissues, respectively, EPA decreased from 7% in protonemata to ~1.3 % in gametophytes. An increase in 20:4- and decrease in 20:5- ethanolamides and their corresponding membrane precursors, phosphatidylethanolamides, also was observed during gametophyte development. Pharmacological studies revealed that AEA specifically inhibits polarized tip growth, which justifies the low endogenous levels of AEA in protonemata. To further determine the physiological relevance of these 20C PU-NAEs, a fatty acid amide hydrolase that catabolizes NAEs has been heterologously characterized. Furthermore, generation of metabolite mutants with altered NAE levels is underway. Overall, we identified two novel NAEs, AEA and EPEA in Physcomitrella, which may play an important role in regulation of moss growth and development, although the underlying mechanism is still unclear.
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Chilufya, Jedaidah, Shiva Devaiah et Aruna Kilaru. « Effects of Anandamide on Development, Growth and Cellular Organization of Physcomitrella Patens ». Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/4798.
Texte intégralRésumé :
Mosses are bryophytes with a simple cellular organization and distinctive growth stages. With their unique lipid profile, most mosses are also tolerant to various stressors. A ubiquitous class of bioactive fatty acid ethanolamides in eukaryotes called N-acylethanolamines (NAEs) also occurs in the moss Physcomitrella patens. Unlike in higher plants, where saturated and unsaturated NAE types are limited to those with acyl chains 12C to 18C, P. patens also contains anandamide, NAE 20:4. In higher plants, NAEs are most abundant in desiccated seeds and mediate plant growth, development, cellular organization and response to stress, in an abscisic acid (ABA)-dependent or independent manner. In mammals, NAE 20:4 acts as an endocannabinoid ligand and mediates a multitude of physiological responses. This unique NAE type, NAE 20:4 is hypothesized to effect development, growth and cellular organization of P. patens. To determine the role of NAEs in moss development, NAE content and composition in protonema, early and late gametophyte stages, and sporophytes, will be quantified from their total lipid extracts, using selective lipidomics. The effects of anandamide on growth will be studied by culturing moss in the presence of exogenous NAE 20:4 in a dose-dependent manner. Temporal changes in growth patterns will be determined by the evaluation of digital images using Image tool. The effect of anandamide on cytoskeletal organization will be visualized by immunostaining the phyllodes exposed to NAE 20:4 and observing them under confocal microscope. Preliminary results indicate that the endogenous NAE content and composition is variable, depending on the developmental stage and that NAE 20:4 is a potent negative regulator of moss growth. More detailed studies are expected to provide novel insights into the role NAEs, specifically NAE 20:4 might play in mediating growth and development of seedless plants.