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SPOROGENESIS AND CALLOSE LOCALIZATION IN ANTHOCERATOPHYTAFlowers, Nicholas David 01 August 2018 (has links)
Spores are fundamental to the reproductive success of all land plants. The success of a spore lies in its recalcitrant multi-layered spore wall commonly made of sporopollenin, cellulose, and pectin. However, other polysaccharides may be associated with the intine of spores, and their patterns of deposition vary across taxa. Callose, a plant 1-3-β-glucan polysaccharide, has unsubstantiated accounts of its presence and absence in association with the spore mother cell wall of hornworts for more than a century. To address this conundrum, I used aniline blue, a fluorochrome that has high specificity of binding to beta glucans and when excited with ultraviolet light, it will fluoresce yellow-green. However, due to the limited resolution power of that technique, I also used transmission electron microscopy (TEM) with immunogold labeling to observe the ultrastructural localization of callose using anticallose, a monoclonal antibody. I also bioinformatically probed the genomes and transcriptomes of hornworts to elucidate the callose synthase genes and enzymes which may be responsible for the putative callose deposition. Because of the asynchronous spore development, each sporophyte has a continuum of spores at various developmental stages. Subsequently, the sporophyte of many hornworts makes an ideal system to study all aspects of sporogenesis. For the first time, I provide unequivocal, correlative evidence for callose involvement in spore wall development in hornworts. Here we report on the spatiotemporal deposition of callose in sporogenesis of Phaeoceros carolinianus, and we show that callose is a common wall constituent of the spore intine in three other genera (Anthoceros, Notothylas, and Nothoceros). Callose deposition in hornworts is post-meiotic and begins during early spore wall development after a white lined lemella is formed and during expansion of the exine. As sporopollenin is deposited and the spore wall thickens, callose remains localized in the intine during the remainder of sporogenesis. The occurrence of callose in hornwort spores is a first record of this polysaccharide in the inner spore wall (intine or endospore) of any embryophyte. This suggests that callose may serve the same or similar roles in hornwort intine development and function as pectin-cellulose does in later diverging taxa. Bioinformatic tblastn techniques and molecular high through put Illumina genome sequencing combined with blast techniques for orthologs to callose synthase genes from Arabidopsis thaliana and Physcomitrella patens did not provide any evidence as to if callose synthase genes are present in hornworts This was due to database contamination, sample contamination, and sample quality.
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Ausgewählte Eigenschaften des Sporopollenins der KieferBohne, Guido 27 February 2007 (has links)
Gegenstand der Arbeit sind Zusammenhänge zwischen physikochemischen Eigenschaften und Funktionen der Exine bei Ausbreitung, Bestäubung und Befruchtung. Dabei bewährte sich der Einsatz der 3-kammrigen Sporopolleninkapseln (Zentralkapsel und Sacci) in der Permeationschromatographie. Sowohl kinetisch bedingte chromatographische Dispersion kleiner Moleküle als auch Konzentrationsänderungen von Zuckern und Dextranmolekülen im Medium wurden zur Bestimmung von Permeabilitätskoeffizienten der Nexine genutzt. Die Wasserabsorptionskapazität von Exinefragmenten und die hydraulische Leitfähigkeit der Nexine wurden anhand von Konzentrationsänderungen ausgeschlossener Dextranmoleküle ermittelt. Das Tectum der saccalen Sexine ist eine Mikrofiltermembran mit scharfer Trenngrenze im Submikrometerbereich; daher werden an den Sacci nur Hydrokolloide mit Stokes''schen Radius über 100 nm (z.B. aus nativem Dextran) ausgeschlossen. Die Nexine ist eine nicht-ideale Umkehrosmose-Membran, die in Zucker- und Salzlösungen hohe Reflexionskoeffizienten zeigt; zusätzlich besitzt sie wenige große Poren, die den Austausch von Zuckern und selbst kleinen Polymermolekülen ermöglichen. Die hydraulische Leitfähigkeit der Nexine liegt im Größenbereich derjenigen von Plasmamembranen (0,39-0,48 µm s-1 MPa-1); die Ergebnisse zeigen, dass die Exine weder die Nährstoffaufnahme des Sporoplasten aus der lokulären Flüssigkeit noch dessen rasche Rehydratation in der Mikropyle behindert. Die Einfaltungen der distalen Nexine (oberhalb der Sacci) und die Omega-Faltung der Exine zwischen den Sacci (Leptom) bieten beim Quellvorgang Schutz vor zu schneller Flächenausdehnung der Plasmamembran. Der Corpus kann mit konzentrierten Elektrolytlösungen beladen werden. Beim anschließenden osmotischen Schwellen in Wasser reißt die Exine, und der Sporoplast wird mit anhaftender Intine ausgeschleudert. Wasser und andere polare Flüssigkeiten adhärieren stärker als hydrophobe Flüssigkeiten an Sporopollenin. Die Sporopolleninmatrix weist eine hohe Feststoffdichte auf, ist wenig quellfähig (0,18 mL g-1 TM) und deformationsstabil. Dies ermöglicht die Pulverbildung beim Trocknen. / Subject of this thesis are relationships between physicochemical properties and functions of the exine concerning propagation, pollination and fecundation. Here the application of the 3-chambered sporopollenin-microcapsules (central capsule and sacci) in permeation chromatography proved of value. Both the kinetically dependent dispersion of small molecules and changes in concentration of sugars and dextran molecules in the medium were analysed to determine permeability coefficients of the nexine. The water absorption capacity of exine fragments and the hydraulic conductance of the nexine were calculated by means of changes in concentrations of excluded dextran molecules. The tectum of the saccal sexine is a microfiltration membrane with a sharp cut off in the submicrometer range; thus hydrocolloids with Stokes´radii over 100 nm (e.g. from native dextran) are excluded from the sacci. The nexine is a non-ideal reverse osmosis membrane having high reflexion coefficients in sugar and salt solutions; in addition few large pores allow the exchange of sugars and even of small polymers. The hydraulic conductance of the nexine is in the range typically for plasmamembranes (0.39-0.48 µm s-1 MPa-1); the results indicate that the exine does neither obstruct the uptake of nutrients by the sporoplast from the locular fluid nor hinder the rapid rehydration in the micropyle. When rehydrating, the distal foldings of the nexine (above the sacci) and the omega-like folding of the exine between the sacci (leptom), provide protection for the plasmamembrane when its surface area has to increase too rapidly. The corpus can be loaded with a concentrated electrolyte solution. When subsequently transferred into water the exine rupture and the sporoplast along with the intact intine is ejected. Water and other polar liquids adhere stronger to sporopollenin than hydrophobic ones. The matrix of sporopollenin show a high density in its solid content, water absorption capacity is low (0.18 mL g-1 DM) and it is resistant to deformation. This enable the formation of powder while dehydrating.
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