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.
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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.
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ItemStudies on the flagella and cytoskeleton of pleurochrysis carterae (prymnesiophycae) and mallomonas splendens (synurophyceae)(University of Melbourne, 1989)The flagellar apparatus of motile, coccolith-bearing cells of Pleurochrysis carterae (Braarud & Fagerlund) Christensen (Prymnesiophyceae) and that of Mallomonas splendens (G.S.West) Playfair (Synurophyceae) are described at interphase and during cell division. The interphase arrangement of the flagellar apparatus in Pleurochrysis carterae is similar to that in Pleurochrysis sp. (Inouye & Pienaar 1985) but I report new information on microtubular root 1 (R1) and describe in detail the transition region of the flagellar axoneme. The basal body of the longer flagellum is shown to be that which is associated with R1. A distinctive membrane decoration is noted on specific areas of the peripheral endoplasmic reticulum (PER) and I discuss the role of the PER in scale secretion and endocytosis. The basal bodies of P. carterae duplicate before mitosis and the crystalline roots (CRs), which arise from R1 and root 2 (R2), disassemble at prophase as their component microtubules elongate towards the future spindle poles. By late prometaphase the basal bodies have segregated semi-conservatively and each pair displays diminutive flagellar roots for the future daughter cells. The two parental basal bodies now bear R1s, indicating that the basal body that produced a short flagellum in the parental cell has transformed into one that will produce a long flagellum in the daughter. All flagellar roots assume their interphase appearance by early cytokinesis. Amputation of the flagella of P. carterae results in their immediate regeneration according to deceleratory kinetics. In the presence of 1?g/ml cycloheximide flagella regenerate to only c. half-length. Immunofluorescence microscopy shows that CRs diminish in size as flagella regenerate and, in the presence of cycloheximide, are depleted when flagella cease to elongate. These data indicate that a pool of presynthesized precursors is available for flagellar regeneration in P. carterae and that part of this pool consists of tubulin from the CRs. Cells of Mallomonas splendens bear a single emergent flagellum (F1) and a non-functional basal body (F2). Direct observations on dividing cells show that the FIs of daughter cells arise from newly formed basal bodies as the parental F1 retracts and thereby transforms into a new F2; the parental F2 remains as such for successive generations. These results are confirmed with thin-sections. The flagellar apparatus of M. splendens at interphase displays numerous features that are novel in the Synurophyceae: the basal bodies are surrounded by a distinctive fibrous capsule; this is continuous with a thick, fibrous band that constitutes the anterior portion of the rhizoplast; the posterior of the rhizoplast forms a cone of numerous, striated straps over the nuclear apex; a single (three-membered) microtubular root (R1) is present which forms a loop around the basal bodies and descends to terminate on the rhizoplast near the nuclear apex; the descending portion of R1 has at least two orientations with respect to the basal bodies. The development of these structures is described from dividing cells. The R1s and rhizoplasts for daughter cells are formed de novo in association with new basal bodies at early mitosis as the parental structures disassemble. The new rhizoplasts are the organizing centres for the mitotic spindle. Mitosis in M. splendens is compared to that in the Chrysophyceae. The final chapter deals with the deployment of the four posterior bristles in M. splendens. Bristles articulate at their flexed basal ends, via an attached fibrillar complex, on specialized body scales (base-plate scales). Bristles are formed independently of their base-plate scales and I describe how they are united outside the cell. Mature posterior bristles are secreted onto the plasma membrane at late interphase and then extruded, basal ends first, from beneath the layer of body scales. Once bristles are fully extruded they are drawn back to the posterior apex of the cell with their basal ends leading - thus a 180� reorientation of the bristles has been effected outside the cell. A cytoplasmic protuberance which contains at least one microtubule accompanies the bristles throughout this reorientation and I propose that it is intimately involved. The fibrillar complex is formed in situ on the bristles and appears to mediate the deployment of bristles onto new base-plate scales.
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ItemAspects of the water relations of eucalyptus behriana F. muell. and E. microcarpa (maiden) maiden near Melton, Victoria(University of Melbourne, 1985)
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ItemStudies on the distribution and cycling of nitrogen in forests(University of Melbourne, 1982)
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ItemPollen-wall proteins and breeding systems of plants(University of Melbourne, 1978)Quatitative cytochemical methods have been developed to estimate changes during development in the pollen-wall proteins. Acid phosphatase was used as a marker for intine proteins, and non specific esterase for the exine proteins. In marrow-stem kale, Brassica oleracea and ryegrass, Lolium perenne the intine enzyme showed two peaks of accumulation during development, the first corresponding to its synthesis and incorporation in the intine, and the second to accumulation in the pollen cytoplasm at maturity. In contrast, the exine enzyme showed a single peak at pollen maturation. This was correlated with dissolution of the tapetum and consequent loss of its very high esterase activity, coincident with the accumulation of esterase in the exine cavities. In sunflower, Helianthus annuus acid phosphatase and esterase were detected in both exine and intine sites during pollen development. Acid phosphatase was associated with the nexine at pre-vacuolate period with high levels in the tapetum. At mid-vacuolate period the tapetum became plasmodial and enzyme activity was detected around the exine surface and was transferred to the exine cavities by the end of the vacuolate period. Esterase activity was associated with the exine at pre-vacuolate period; subsequently during the vacuolate period, activity was present in the intine, and in the cytoplasm at maturity. Two peaks of accumulation were detected, closely resembling those for the intine marker enzyme in Brassica and Lolium. The developmental cytochemistry of the stigma of Helianthus, Lolium and Secale has been investigated with a view to understanding the nature of selfincompatibility, the site of tube arrest and the route of pollen tube penetration. The callose rejection response in pollen and stigma that has been established for Cosmos and various Crucifers have been found in Helianthus. The rejection reaction is found to occur in the pollen grain and pollen tubes of grasses. The callose produced in germinating self pollen of the grass, Secale cereale has been isolated by a degredative physicochemical procedure. Partial acid hydrolysis, enzyme hydrolysis, sugar analysis and methylation analysis have shown that the material is a 1,3-?-linked glucan; however some evidence points to the presence of 1,4-?-linkages in addition.
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ItemThe ecology of amsinckia (amsinckia spp.) in wheat crops in Victoria(University of Melbourne, 1977)Amsinckia hispida, A. lycopsoides and A. intermedia are serious annual weeds of wheat crops in Victoria. Studies of cytology, seed germination, reproductive development, growth physiology, and competitive ability in relation to wheat were undertaken to elucidate the biological and ecological characteristics of Amsinckia that contribute to the success of these species as weeds. The most widespread species in Victoria is A. hispida, which was used in all the experimental studies. Observations in the field included all three species. Chromosome counts indicated the close relationship between A. hispida, a native of South America, and the other two North American species. All present similar problems as weeds. Germination was found to be regulated by a primary dormancy, which confined germination during after-ripening to low-temperatures (<10�C) and short photoperiods (darkness to 8 hr). In the field germination occurred as a flush, the magnitude of which depended on the timing and amount of rainfall, and the depth of burial. Seed produced in the previous spring, lying on or close to the soil surface, gave high germination percentages from the autumn to early winter. Ungerminated buried seed developed a light requirement for germination, which may be satisfied by cultivation. Seed held in the dark for 56 days at temperatures of 15� and 25�C developed a secondary dormancy, which greatly restricted germination under normally favourable conditions. These regulatory mechanisms would serve to confine germination to the normal growing season for Amsinckia, while providing several strategies for coping with adverse seasonal conditions as well as periodic cultivation. Reproductive development to flower initiation was retarded by increase in temperature from 10* to 20�C, whereas subsequent development was hastened by the same temperature increase. Increase in photoperiod from 8 to 16 hr greatly hastened development to flowering. Environmental regulation of development ensures that Amsinckia plants complete their life cycle before unfavourable conditions ensue in the late spring, irrespective of the time of germination, while balancing vegetative growth for maximum effectiveness in competition with reproductive growth for maximum seed production. Vegetative growth was characterized by a high relative growth rate (c. 0.18 mg mg-1 day-1) under moderate temperatures (15-20�C and high irradiance (no shade), associated with a high net assimilation rate (0.4-0.5 mg cm-2 day-1 ) and a high leaf area ratio (0.3-0.4 cm2 mg-1). A temperature optimum for growth in the range 15-20�C found in Amsinckia, is amongst the lowest reported for a temperate species. Potential seed production was extremely high - up to 2,000 seeds per plant in sward conditions. In competition with wheat, Amsinckia depended on its ability to respond rapidly to changes in the aerial and soil environments, in order that it maintain its share of light, nutrients and moisture. A reduction in irradiance elicited increases in leaf angle, leaf and stem lengths, specific leaf area, and shoot to root ratio. A reduction in the supply of soil nutrients resulted in a decrease in the shoot to root ratio. Where there was intense competition for both light and nutrients Amsinckia shoot dry weight was reduced by about 30%. Reductions in the yield of wheat resulting from competition from Amsinckia may be associated with a reduced supply of light,nutrients and/or moisture, but the results suggest that competition for soil moisture may have the greatest effect on wheat yields. The results have important implications on methods used to control Amsinckia. The possibility of reducing the effectiveness of Amsinckia by encouraging competition from wheat in the crop is stressed.