School of Botany - Theses
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ItemPeriplast structure and development in the Cryptophyceae(University of Melbourne, 1994)The distinct asymmetric shape of cryptomonad cells is maintained by a complex organelle termed the periplast, which consists of a plasma-membrane (PM) and two additional layers, the inner periplast component (IPC) and surface periplast component (SPC). In this study, periplast morphology is examined in representatives of fifteen cryptomonad genera using a combination of electron microscope techniques. Scanning electron microscopy, thin sections and freeze-fracture/-etch allow detailed examination of the IPC, PM and SPC, and reveal an intimate relationship between periplast components in many cryptomonad genera. Differences in periplast morphology (combined with variations in periplast arrangement across cells) enable recognition of eleven periplast types within the Cryptophyceae. The IPC (which forms the primary structural element of the periplast) may consist of a continuous sheet of material, or comprise an ordered system of discrete plates. In numerous genera, the IPC is closely appressed to (and intimately associated with) the PM. Freeze-fracture images of the PM reveal discrete domains, densely packed with intramembrane particles (IMPs) wherever the IPC and PM are in direct contact. In contrast, regions of PM not supported by the IPC appear less ordered, and generally contain fewer IMPs. These observations suggest that the IPC may act as a template which influences organization of IMPs within the PM. Freeze-fracture/ etch also enables detailed examination of SPC microarchitecture. Surface structures range from dense mats of randomly arranged fibrils and scales to complex crystalline plates. Crystalline surface plates are always positioned directly �above� ordered PM domains, suggesting an intimate relationship between organization of the SPC, PM and IPC in many cryptomonads. Despite variations in periplast morphology throughout the Cryptophyceae, similarities in cytokinesis and periplast development are evident in a wide range of genera. In all cryptomonads examined in this study, cell division involves a unique process termed �pole reversal�. During cytokinesis the tail regions of daughter cells develop from the anterior of the parental cell, necessitating complete realignment of the periplast. Following cell division, daughter cells are smaller than the parental cell, and considerable periplast development occurs as cells enlarge and mature. Detailed examination of cryptomonads possessing an IPC of discrete inner plates indicates that orderly growth of the periplast occurs from specialized regions termed anamorphic zones. Inner plates form de novo (and undergo substantial enlargement) within anamorphic zones throughout the cell cycle. Freeze-etch examination suggests that crystalline surface plates also develop within anamorphic zones, by self-assembly of disordered subunits on the cell surface. The close relationship between SPC, PM and IPC suggests that incorporation of disordered subunits into the crystalline surface plates may be associated with growth of the inner plates and PM domains. The complex surface scales found in a wide range of cryptomonads also appear to form on the cell surface via self-assembly of less ordered precursors. Development of surface scales, however, does not appear linked to changes in the underlying IPC and PM.
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ItemThe extracellular matrix and cell walls of pistils of Nicotiana alata(University of Melbourne, 1994)Pistils of the reproductive tissues of flowering plants comprise the stigma, style and ovary. During pollination, pollen grains germinate on the stigma and produce pollen tubes that grow extracellularly through the transmitting tissue of the style to the ovary, where they effect fertilisation. The transmitting-tissue cells secrete an extracellular matrix through which pollen tubes grow, and thus transmitting-tissue cell walls and the secreted components of the extracellular matrix are important in the process of fertilisation. Cell walls of styles of Nicotiana alata Link et Otto (ornamental tobacco) were analysed chemically and examined histochemically. The stylar epidermal cells were shown histochemically to have thick, lignified secondary walls. These walls probably constituted a large proportion of cell-wall preparations from whole styles because analysis of whole style walls indicated that the major polysaccharides present were xylans and cellulose, which are typical of lignified secondary walls of dicotyledons. Analysis of cell- wall preparations from isolated transmitting-tissue cells were different, indicating that these contained cellulose, xyloglucans, and pectic polysaccharides, which are typical of primary cell walls of dicotyledons. However, the analysis indicated that the walls also contained an unusually high proportion of arabinogalactan proteins (AGPs). Staining of the transmitting-tissue cell-wall preparation with ?-glucosyl Yariv reagent, a histochemical reagent specific for AGPs, confirmed their presence in these walls, which may be related to the role of these cells in secreting the stylar extracellular matrix. AGPs from the pistils of N. alata were found to be developmentally regulated, as the different charge classes of AGPs altered during floral development. The AGPs from the mature stigma, style and ovary showed distinct charge characteristics. Both the amount and concentration of AGP in the stigma increased markedly between petal colouration and maturity, and continued to increase up to 48 h post-maturity. The concentration of AGP in the style and ovary remained almost constant throughout development, however, and the amount of AGP in these organs increased only in proportion to their fresh weight. Following pollination there was an increase in the amount of AGPs in the stigma, and this increase was independent of the self-incompatibility genotype of the pollen. Staining of the pistil with ?-glucosyl Yariv reagent demonstrated the presence of AGPs on the stigma surface, throughout the transmitting tract, and on the epidermis of the placenta which connects with the ovules. AGPs from stigmas and styles of N. alata were purified by affinity chromatography using a monoclonal antibody (J539) linked to Sepharose 4B, or by selective precipitation using ?-glucosyl Yariv reagent. The AGPs were purified by gel filtration chromatography under dissociating conditions. The purified AGPs were shown to have characteristics typical of other AGPs, and contained a high proportion of carbohydrate (>90%), with a high ratio of galactose to arabinose (2:1). The protein content was approximately 5%, and contained high levels of alanine, serine and hydroxyproline. The AGPs consisted of a major species which was almost neutral and a minor species which was more negatively charged. Sedimentation equilibrium experiments showed that the purified AGPs had a mean molecular weight of 143 kilodaltons. AGPs isolated from pistils of plants of self-incompatibility genotypes S2S2 and S6S6 were similar with respect to monosaccharide composition, amino-acid composition, charge and molecular weight. Linkage analysis showed that the purified AGPs contained a highly branched backbone of 3-, 6- and 3,6-linked galactopyranose residues, bearing terminal galactopyranose and terminal arabinofuranose residues. Analysis by one-dimensional and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy confirmed the presence of these glycosyl linkage types, and showed a high mobility of the terminal arabinofuranose residues consistent with their location on the periphery of the molecules. This analysis represents the first full assignments for AGP molecules in solution. No difference was found between AGPs purified by affinity chromatography or selective precipitation, between AGPs purified separately from stigmatic or stylar tissue, or between AGPs purified from pistils of plants of different self-incompatibility genotypes.
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ItemThe production of foreign peptides and proteins in plant-cell culture(University of Melbourne, 1994)There is growing demand from medicine, industry and agriculture for large quantities of purified peptides and proteins. This thesis describes the use of cultured plant cells for the production of peptides and proteins from gene constructs introduced by transformation. The culture is a Nicotiana plumbaginifolia cell line (NPT5120) grown as callus and as suspension-cell cultures. Initially we were interested in the expression of analogues (denoted AVPA, AVP-G) of the mammalian peptide hormone arginine vasopressin (AVP). Peptide was secreted by the transformed cells but was unstable in the culture and did not accumulate to high levels (-77 pg/20 ml culture). This problem was partially solved by the addition of the protease inhibitor, bacitracin, however, bacitracin did not completely prevent the loss of the peptide and it retarded plant cell growth. In a further attempt to stabilize the peptide, it was incorporated within the S2-RNase from N. alata because the S2-RNase is stable in the extracellular environment of pistils, and in the presence of protein extracts from the plant-cell cultures. The S-RNases of solanaceous species have a hypervariable region which was replaced with the peptide analogue in these studies. The transformed plant cells produced a transcript (-940 bases) encoding the hybrid RNase at a level 30-fold lower than the level of transcript encoding the S2-RNase in styles of N. alata. Although hybrid transcript was detected readily, the hybrid-RNase was not detected, indicating that the modification at the hypervariable region was not tolerated. A similar result was obtained when the cells were transformed with a construct encoding the mature protein from the major house dust mite allergen (Der p 1) from Dermatophagoides pteronyssinus. The allergen did not accumulate in the plant culture even though transcript encoding Der p 1 was detected. It is likely that translation and/or post-translational processing was inefficient or absent. In contrast, when the cell line was used for the expression of an unmodified plant protein, the proteinase inhibitor (PI) from N. alata, PI protein accumulated in the transformed cells to detectable levels, -0.01% of the total protein. Thus the N. plumbaginifolia plant-cell culture has potential for the expression of foreign peptides and proteins but a better understanding of translation, co- and post-translational processes together with product stability is required before this system can be used to produce large quantities of foreign peptides and proteins.