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ItemNo Preview AvailableFeeding behaviour in mixotrophic chrysophytes : qualitative and quantitative observations using video microscopy(University of Melbourne, 1999)Complexity in the natural history and feeding behaviour of mixotrophic chrysophytes is investigated using video microscopy and digital image-enhanced observations of living cells. An introductory survey of eight species of freshwater chrysophytes demonstrates variability in characteristics of the natural history of cells in culture and suggests a diversity of mixotrophic strategies, or balance between photosynthesis and phagotrophy for nutrition, in the species examined. Four levels of phagotrophic dependence are suggested based on variability in polymorphic life cycles, feeding mechanisms, continuous or bimodal (on/off) feeding in the trophic stage, associations of chrysophytes with bacteria and general growth and pigment characters. Two feeding-types are reported: (i) the Ochromonas-like stage that feeds by a direct interception, or raptorial, mechanism occurs in seven out of the eight species examined (Ochromonas spp., Poterioochromonas spp., Chromulina nebulosa, Lepochromulina bursa, Epipyxis pulchra), and (ii) Chrysamoeba mikrokonta is a rhizopodial chrysophyte that feeds by diffusion. Qualitative multi-step feeding models, which include active (post-encounter) and passive stages, are proposed for both mechanisms and are compared to the classical direct interception and diffusion concepts. Feeding behaviour in a sessile Ochromonas-like chrysophyte, Epipyxis pulchra, is investigated in more detail using a flow-chamber system Size-selective feeding is studied by offering Epipyxis pulchra cells beads of various sizes and describing the observed responses in the context of the multi-step feeding model. The observations demonstrate that both passive and active mechanisms delineate the ingestable size-range, and the preferred sizes within this range. Subsequently, feeding-history- related-selection (FHRS), or active selection behaviour that is triggered by factors that are related to the recent feeding history of cells, is investigated. In Epipyxis pulchra, cells that are photrophically grown and never previously exposed to beads, the retention efficiency on preferred bead sizes (0.87 ?m) is high (c. 100%), but cells tend to reject beads as their recent feeding history expands. The outcome of encounters with beads in cells with variable recent feeding histories is analysed using logistic regression modelling and hypotheses that could explain the underlying biological mechanism(s) of FHRS are discussed. A further complexity in the feeding strategy of Epipyxis pulchra, is its bimodal feeding behaviour. The available evidence demonstrates that the behavioural strategies, or the combined effects of active particle rejection and non-continuous feeding, can vary when Epipyxis pulchra cells are offered different bacteria species and when different prey concentrations are available. Overall, there is a tendency for cells to spend more time switched-off when offered bacteria in the flow-chamber than when cells are offered beads in the same conditions (P < 0.001). The behavioural complexity demonstrated in Epipyxis pulchra provides a perspective for explaining variable feeding strategies among direct interception feeding chrysophytes. Some overall ecological implications of flexible behaviour and life cycles in mixotrophic chrysophytes are discussed in relation to the role of mixotrophic chrysophytes in microbial food webs and for the interpretation of grazing experiments that are dependent on simple models of predation.
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ItemNo Preview AvailableFeeding behaviour in mixotrophic chrysophytes : qualitative and quantitative observations using video microscopy(University of Melbourne, 1999)Complexity in the natural history and feeding behaviour of mixotrophic chrysophytes is investigated using video microscopy and digital image-enhanced observations of living cells. An introductory survey of eight species of freshwater chrysophytes demonstrates variability in characteristics of the natural history of cells in culture and suggests a diversity of mixotrophic strategies, or balance between photosynthesis and phagotrophy for nutrition, in the species examined. Four levels of phagotrophic dependence are suggested based on variability in polymorphic life cycles, feeding mechanisms, continuous or bimodal (on/off) feeding in the trophic stage, associations of chrysophytes with bacteria and general growth and pigment characters. Two feeding-types are reported: (i) the Ochromonas-like stage that feeds by a direct interception, or raptorial, mechanism occurs in seven out of the eight species examined (Ochromonas spp., Poterioochromonas spp., Chromulina nebulosa, Lepochromulina bursa, Epipyxis pulchra), and (ii) Chrysamoeba mikrokonta is a rhizopodial chrysophyte that feeds by diffusion. Qualitative multi-step feeding models, which include active (post-encounter) and passive stages, are proposed for both mechanisms and are compared to the classical direct interception and diffusion concepts. Feeding behaviour in a sessile Ochromonas-like chrysophyte, Epipyxis pulchra, is investigated in more detail using a flow-chamber system Size-selective feeding is studied by offering Epipyxis pulchra cells beads of various sizes and describing the observed responses in the context of the multi-step feeding model. The observations demonstrate that both passive and active mechanisms delineate the ingestable size-range, and the preferred sizes within this range. Subsequently, feeding-history- related-selection (FHRS), or active selection behaviour that is triggered by factors that are related to the recent feeding history of cells, is investigated. In Epipyxis pulchra, cells that are photrophically grown and never previously exposed to beads, the retention efficiency on preferred bead sizes (0.87 ?m) is high (c. 100%), but cells tend to reject beads as their recent feeding history expands. The outcome of encounters with beads in cells with variable recent feeding histories is analysed using logistic regression modelling and hypotheses that could explain the underlying biological mechanism(s) of FHRS are discussed. A further complexity in the feeding strategy of Epipyxis pulchra, is its bimodal feeding behaviour. The available evidence demonstrates that the behavioural strategies, or the combined effects of active particle rejection and non-continuous feeding, can vary when Epipyxis pulchra cells are offered different bacteria species and when different prey concentrations are available. Overall, there is a tendency for cells to spend more time switched-off when offered bacteria in the flow-chamber than when cells are offered beads in the same conditions (P < 0.001). The behavioural complexity demonstrated in Epipyxis pulchra provides a perspective for explaining variable feeding strategies among direct interception feeding chrysophytes. Some overall ecological implications of flexible behaviour and life cycles in mixotrophic chrysophytes are discussed in relation to the role of mixotrophic chrysophytes in microbial food webs and for the interpretation of grazing experiments that are dependent on simple models of predation.
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ItemAustralian red algae of the families Solieriaceae and Cystocloniaceae : cell wall polysaccharides and phylogenetic assessment(University of Melbourne, 1997)The cell wall polysaccharides extracted with hot water from Australian red algae of the family Solieriaceae (Gigartinales) were characterised by chemical and spectroscopic techniques. The polysaccharides from six species of the genus Callophycus were sulfated (15.9 - 16.8% w/w) galactans containing the highest levels of pyruvate so far reported for red algal polysaccharides (8 - 10.5% w/w). Evidence from 13C NMR spectroscopy demonstrated that the galactans were carrageenans rather than agar-type polysaccharides. Characterisation of the galactans with Fourier transform infrared (FTIR) spectroscopy, linkage analyses, and ID and 2D nuclear magnetic resonance (NMR) spectroscopy demonstrated they were comprised predominantly of the unusual, pyruvated repeating disaccharide 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate, and smaller amounts of carrabiose 2-sulfate (the repeating unit of a-carrageenan) and other structural variations. Detailed characterisation of the Callophycus carrageenans enabled confirmation by 13C NMR spectroscopy of the presence of pyruvated carrabiose 2-sulfate in the galactans from other species. For example, the carrageenan from Sarconema filiforme was composed of a hybrid or mixture of approximately equal proportions of the repeating units of ?-carrageenan, ?-carrageenan, and 4',6�-O-(1-carboxyethylidene)carrabiose 2-sulfate. Carrageenan from Solieria robusta was composed predominantly of ?-carrageenan, with a minor component of 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate. In addition, the constituent sugars and FTIR spectrum of the galactan extracted from Tikvahiella candida, an adelphoparasite of Solieria robusta, resembled those of the carrageenan from the host. The polysaccharides from Rhabdonia coccinea and R. verticillata gave FTIR spectra resembling that of ?-carrageenan, but were rich in 6-O-methylgalactose (6-MeGal; ca. 31 mol% and 17 mol% of constituent sugars for R. coccinea and R. verticillata, respectively). Data from 13C NMR spectroscopy showed that the polysaccharides were carrageenans rather than agars. The preparations contained mainly carrabiose 2,4'- disulfate of ?-carrageenan, partially methylated at 0-6 of the 3-linked residues. In addition, the polysaccharide from R. coccinea contained small quantities of ?-carrageenan structure, whereas the polysaccharide from R. verticillata contained some 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate and 3-O-methylgalactose (3-MeGal). The unusual sugar 3-MeGal occurred primarily as main-chain 4-linked residues, with a small proportion in the form of terminal residues. The carrageenans from three species of Erythroclonium were shown to be highly substituted carrageenans with at least five types of repeating disaccharide units, including those of ?- and ?-carrageenans, their 6'-0-methylated counterparts, and 4',6'-O-(1-carboxyethylidene) carrabiose 2-sulfate. These polysaccharides also contained significant quantities of unsubstituted, 4-linked Galp and small quantities of 4-linked 3-MeGalg and terminal glycosyl residues. The similarities in MeGal content (> 20 mol% of constituent sugars), sulfation pattern, pyruvate acetal substitution, and occurrence of 4-linked Galp and 3-MeGalp residues in the carrageenans of Rhabdonia, Erythroclonium, and Areschougia support a close relationship between the three genera. Interestingly, Melanema dumosum, which was previously classified as an Areschougia species, produces a hybrid or mixture of the repeating units of ?, ?- and ?-carrageenans essentially devoid of MeGal and bearing little resemblance to those from Rhabdonia, Erythroclonium, and Areschougia spp., indicating that Melanema may not be closely related to the other three genera. A survey of the galactans from eight species representing six genera of the family Cystocloniaceae indicated that most species essentially produce ?-carrageenan, although some minor variations in structure occur. One anomalous species was Austroclonium charoides (previously classified as a Rhabdonia species), which produced carrageenans with high levels of MeGal (21 mol% of constituent sugars) and sulfation patterns resembling those of the carrageenans from Rhabdonia, Erythroclonium, and Areschougia. Together with observations of morphology, anatomy, and reproductive development of Austroclonium charoides, which also suggest the alga is more closely related to Rhabdonia, Erythroclonium, and Areschougia spp. than it is to other members of the Cystocloniaceae, the carbohydrate data supported the provisional transfer of Austroclonium charoides to the Solieriaceae. Phylogenetic analyses of SSU sequences of representatives of the families Solieriaceae, Cystocloniaceae, and Caulacanthaceae, as well as those of other families in the Gigartinales, supported the hypothesis that Austroclonium charoides was misclassified in the family Cystocloniaceae. A. charoides allied instead with Rhabdonia verticillata, and the remaining representatives of the Cystocloniaceae formed a well-resolved, strongly supported monophyletic group. The family Solieriaceae is apparently polyphyletic, with representatives of Kappaphycus, Eucheuma, Betaphycus, and Solieria resolving as a monophyletic group to the exclusion of representatives of Rhabdonia, Erythroclonium, Areschougia, and Callophycus. The latter four genera, with Austroclonium, were only weakly allied, however. Although these data give precedence to resurrecting the Rhabdoniaceae (as Areschougiaceae), the problems of determining the likely generic composition of the family are discussed.
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ItemOntogeny and morphology of flowers and inflorescences of Nothofagus Bl. (Southern beeches)(University of Melbourne, 1996)Organogenesis of pistillate and staminate inflorescences in selected species of Nothofagus is described along with the comparative morphology of mature flowers. Staminate flowers in the subgenera Brassospora, Nothofagus, and Fuscospora have less than 20 stamens, with three to five, usually four tepals in each flower. Organogenetic studies demonstrated that the flowers in subgenera Brassospora and Fuscospora exhibit centripetal androecium development. The staminate inflorescences in these subgenera often consist of three flowers and the temporal pattern of floral development, and arrangement of subtending prophylls, support interpretation of the inflorescence as a dichasium. Lophozonia differs from the other subgenera in that the staminate 'flower' is a solitary structure with large numbers of stamens (up to 60 in some taxa) and broadly campanulate perianth with usually 8-12 lobes. Centrifugal development occurs in all taxa studied and the solitary 'flower' in Lophozonia is interpreted as a fused dichasium, a pseudanthium. The fused dichasium is unique to subgenus Lophozonia and defines its species as monophyletic. Pistillate inflorescence organogenesis in subgenera Lophozonia, Fuscospora and Nothofagus is described. The temporal pattern of development, and the arrangement of flowers, prophylls and cupule valves in the pistillate inflorescences, is shown to be comparable in all taxa. While the cupules in Brassospora remain inadequately studied, the architecture of the valves and fruits can similarly be interpreted as a having a cymose architecture. The cupules of all Nothofagus species can be interpreted as having a variously modified cymose structure. The cupule valves occur in the axil of a bract and can be interpreted as an modified shoot system. Previous interpretations of the cupule of Nothofagus as either an intercalary structure (van Steenis, 1953), an organ sui generis (Forman, 1966a), or a modified perianth (Jenkins, 1993) are shown to be inconsistent with the cupule morphology revealed in this study. Organogenetic studies of the cupules in different subgenera demonstrated that the appendages develop acropetally and have a similar ontogeny. The architecture and arrangement of appendages on the cupule valves in the subgenera Nothofagus, Fuscospora and Lophozonia is fundamentally the same in all species studied. Appendages on the cupules in Nothofagus are arranged in regular horizontal rows. The free (not united) laciniate appendages of Lophozonia, and also of some taxa in subgenus Nothofagus, are considered homologous with the lamellae in Fuscospora which are initiated as separate structures but fuse to form continuous horizontal units. Using Nelson's (1978) reformulated Biogenetic law that "given an ontogenetic character transformation, from a character observed to be to be more general to a character observed to be less general, the more general character is primitive and the less general advanced" then the lamellae of the subgenus Fuscospora (the less general condition) are considered derived. The pistillate inflorescences in Nothofagus and Fagacene are interpreted as being structurally homologous. The cupules in both taxa are axillary structures, and the fruits and valves exhibit a cymose architecture. The character 'cupule' is considered plesiomorphic in both families. The architecture of the cupule valve in Nothofagus can be interpreted as either an entirely sympodial system, forming a rhipidium/drepanium-structure, or alternatively a monopodial system incorporating cymose components. Both hypotheses differ to that proposed for Fagus and Castanea by Fey and Endress (1983). The architecture of the cupule valves, and the arrangement of appendages in each valve, however, differ between Fagus/Castanea and Nothofagus.
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ItemDNA phylogeny of eucalypts(University of Melbourne, 1995)Eucalyptus is the dominant tree of Australia's forests and woodlands and is of considerable economic importance, so substantial efforts have been made by many researchers to discover the phylogenetic relationships within this large genus. Based on previous studies of morphological characters, Eucalyptus may be either monophyletic or polyphyletic. The rapid growth of molecular biology in the last ten years has made it practical to sequence DNA, from a number of taxa, for use as characters in phylogenetic analyses. This project involved sequencing the 5S rDNA repeat from four outgroup taxa (Arillastrum group), two Angophora species and twenty Eucalyptus species (including representatives from all informal subgenera). Alignment shows that, as in other plants, the tandemly repeated 5S rRNA genes are highly conserved whilst the non-coding intergenic spacers are variable. A 50 base pair repeating element, which has undergone duplication and modification in certain taxa, is identifiable within the spacer and accounts for much of the variability. There were several other multi-residue deletion/insertion events in the spacers that define major clades. Southern hybridisation and PCR experiments suggest that these different 5S rDNA repeat units are orthologous and hence, suitable as markers of the phylogenetic history of the taxa from which they were derived. Based on the indel events and modifications of the 50 bp element, and parsimony analysis of the sequence data, bloodwood subgenera, Corymbia and Blakella, are shown related to Angophora rather than to non-bloodwoods, Eudesmia, Idiogenes, Gaubaea, Monocalyptus, Telocalyptus and Symphyomyrtus. Relationships within the non-bloodwood groups, however, were not resolved by the 5S rDNA data. In addition to analysis of the 5S rDNA data set, published chloroplast DNA RFLPs and a morphological data set were reanalysed and found to be informative at different levels of the eucalypt clade. Major subclades were analysed separately, the results of which, when combined, gave a single/ phylogenetic tree for Angophora and Eucalyptus. The phylogenetic hypothesis suggests interpretations for homoplasious morphological characters, including parallel evolution of sepaline and petaline opercula (and associated stemonophore) and types of conflorescence. Ovule and seed characters proved to be most informative in the morphological data set. For taxonomic revision, the tree shows that Eucalyptus is paraphyletic and supports the recognition of bloodwood eucalypts as a new genus. Although together Corymbia and Blakella are, monophyletic, Corymbia itself is paraphyletic. The tree supports recognition of three major clades within the non-bloodwood eucalypts ("eudesmids", "symphyomyrts" and "monocalypts") and suggests relationships for taxa within each of these.
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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.
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ItemBiomass, growth and nutrient cycling in regenerating eucalyptus regnans forest after fire(University of Melbourne, 1992)The main theme of this thesis includes two aspects: (1) the biomass, growth, productivity and nutrient cycling in the regenerating Eucalyptus regnans F. Muell (Mountain Ash) forest at Britannia Creek after fire, and (2) to test the hypothesis that nutrient losses due to fire might lead to a decline in stand productivity. The studies in this thesis were based in the Britannia Creek Experimental Forest (Attiwill 1991a), which is a regenerating E. regnans forest on a logging coupe burned in the 1983 (Ash Wednesday) wild-fires. A total of 31 trees along the diameter range was harvested over four years from 1987 to estimate biomass and nutrient content. Monthly litter collections were made in the same period from 24 plots with different fertilizer treatments to assess changes in litterfall mass and nutrient returns. Fortnightly soil samples were taken to examine the impacts of fertilizer on nutrient availability, transformation and plant uptake. E. regnans is a fire-climax species, and seedlings regenerate in profusion after fire. Thirty-nine months after regeneration started there were 16700 stems ha-1, but the stocking density declined rapidly with an annual mortality rate of about 31% in numbers during study period. The annual growth of biomass, however, was 23 tonnes ha-1, an increase rate of 33% on average. By the end of 1989, at age 6.25 years, the forest accumulated 118 tonnes ha-1 in above-ground biomass of eucalypt trees, and 274 m3 ha-1 in volume. Nutrient contents in biomass increased with forest growth. At age 6.25, the above-ground biomass contained 294 kg ha-1 of N, 25 kg ha- 1 of P, 311 kg ha-1 of K, 239 kg ha-1 of Ca, and 79 kg ha-1 of Mg. Litterfall of 5.7 to 9.2 tonnes ha-1 year-1 is at the upper end of the range for eucalypt forests. The proportion of dead eucalypt leaves in litterfall is also large. Therefore, litterfall returned substantial amounts of nutrients back to the forest floor. The amounts returned in litterfall for N, P, K, Ca, and Mg are 78, 4, 16, 61, and 13 kg ha-1 year-1 respectively in 1989. About 48% and 34% of the gross annual demands for N (126 kg ha-1 year-1) and for P (9 kg ha-1 year-1) were met by annual litter return and a little less than 20% by internal redistribution, although the demand for minerals from soil reserves was still large. The net primary production (NPP) of 45 tonnes ha-1 year-1 (eucalypt trees alone) made this forest the most productive among any natural forests, including tropical forests, reported so far. There is certainly no evidence of decline in productivity following clearfelling and burning. The addition of fertilizers temporarily increased the availability of applied nutrients. However, the increased concentration of available N returned to pre-addition level two months after fertilizer application, while the increased concentration of P was sustained for at least two years. Net N-immobilization occurred immediately after N fertilizer application, and it is apparently the most important process in the conservation of N fertilizer. A total of 24 kg ha-1 of the N, 11% of N added, was immobilized in the surface 5 cm of soil in the second month alone after fertilizer application. Only a small proportion (5.5%) of added N was taken up by plants. Although greater concentrations of added nutrients were found in dead eucalypt leaves falling as litter, there was no difference in concentration in live leaves across the different fertilizer treatments. The additions of up to 1000 kg ha-1 of N and 500 kg ha-1 of P, together with all other essential elements, have not increased either biomass or productivity in the E . regnans forest so far. The E . regnans forest at Britannia Creek is therefore not limited by N or P or any other essential nutrients, and losses of nutrients due to fire have not resulted in a decline in productivity of the subsequent forest.
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ItemCharacterization of pistil-specific genes encoding proline-rich proteins from Nicotiana alata(University of Melbourne, 1990)This thesis describes investigations into the structure and expression of a group of pistil-specific genes encoding proline-rich proteins in Nicotiana alata. Two cDNA libraries prepared from pistil mRNA of N. alata were screened with a probe encoding a carrot extensin. Low stringency hybridization and washing conditions were used to enhance the chance of detecting DNA sequences encoding high proportions of proline residues but which might be distinct from extensin. Three classes of cDNA clones, corresponding to three proline-rich protein genes (PRP1, PRP2 and PRP3), were isolated from the pistil cDNA libraries. None of these cDNA clones represented a full-length transcript Two genomic clones (PRP3g5 and PRP3g12) corresponding to the PRP3 gene were also characterized. The PRP3g5 clone probably represents a pseudogene of the PRP3 gene family, whereas the clone PRP3g12 is likely to correspond to a functional PRP3 gene. A typical TATA box and several putative polyadenylation signal are present in the sequence of the clone PRP3g12, and the ATG initiation codon of this gene is found to be located in a context which is probably optimum for translation initiation. The clone PRP3g12 does not contain any introns. The three proline-rich proteins predicted from the partial cDNA clones and the genomic clone are all rich in proline residues (29% for PRP1,49% for PRP2 and 30% for PRP3). In addition, PRP1 is also rich in asparagine (14%), and PRP3 is rich in serine (19%). These three genes have a similar codon usage preference for praline: more than 60% of all proline residues are coded by CCA. The 114 amino acid sequence of PRP2 can be divided into two domains; one (nucleotides 1-210) contains 5 repeats of (Pro)4-Ala interspersed with 4 repeats of the pentapeptide Gln-Leu-Pro-Ile-Arg, and the other is composed mainly of tandemly reiterated (Pro)2-5-Gly-Tyr repeats. The first 23 of the 151 amino acid residues in the PRP3 protein predicted from the genomic clone PRP3g12 are hydrophobic and resemble to a signal peptide. The PRP3 resembles extensin in containing six Ser-(Pro)4 repeats which are separated, in most cases, by a Ser-Pro dipeptide. Southern analysis under stringent conditions showed each of the three PRP cDNAs hybridized to a number of restriction fragments for each of the four restriction enzymes used. This indicates that each of these PRP genes belongs to a small multigene family. The carrot extensin genomic clone (pDCSA1) hybridized to a set of restriction fragments different from any of the three PRP genes, suggesting that another gene more homologous to the carrot extensin than any of the three PRP genes is present in N. alata. The hybridization pattern of the clone pDC5A1 is relatively simple, indicating a low copy number of the extensin gene in the genome. Northern analysis indicated that the three PRP genes are different from each other and from the extensin gene. All the three PRP genes are effectively pistil- specific, in contrast to the extensin gene which is expressed in all tissues tested although the level, number and size of the extensin transcripts differs in different tissues of N. alata. The PRP3 gene is developmentally regulated and its maximum expression correlates with the maturity of the pistil. After wounding, the expression of the extensin gene is increased in all tissues tested, whereas, the mRNA levels of both PRP2 and PRP3 decreased in wounded pistils. Wounding had no detectable effect on the expression of the PRP3 gene in stem and leaf tissues, while a small PRP2 transcript (smaller than the major transcript expressed in wounded pistils) is induced in the same tissues after wounding.