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Coccolithophores are single-celled marine phytoplankton, which produce intricate calcium carbonate platelets or ‘coccoliths’. Emiliania huxleyi is the most abundant and widespread coccolithophore, and is one of the most productive calcifying species on earth, playing a key role in global carbon, carbonate and sulphur cycles. Despite much research into coccolithophore biology, the underlying function of their coccoliths is still unknown. The main aim of the research reported in this thesis was to examine the impact of calcification on metabolism in coccolithophores. Calcification is a significant global process, so it is important to discover what effect it has on the metabolism of cells. The major metabolites each have different costs and benefits to the cell, which will vary depending on the habitat and environmental conditions the cell is in. By comparing the metabolite profiles of different strains, including calcifying, non-calcifying, haploid and diploid cells, differences in metabolite composition and potential patterns related to cell type were investigated. Low molecular weight (LMW) metabolites were characterised using a combination of metabolomic techniques. In agreement with previous research, dimethylsulphoniopropionate (DMSP) was the most abundant compound, followed by mannitol and glycine betaine (GBT). Less abundant sugars, polyols and amino acids were also identified. Environmental factors were manipulated to investigate how the principal metabolites were affected by salinity, different light intensities and nutrient (phosphate and nitrate) limitation. The data revealed a striking difference between haploid and diploid cells of the same strain, with the haploid containing lower concentrations of most of the major metabolites. Thus it is proposed that haploid cells have a different osmoregulatory strategy from the diploid cells. A negative correlation was found between DMSP and mannitol, suggesting that mannitol has a dual function, not only as a major storage compound but also as a principal compatible solute. Untargeted metabolite profiling is becoming a popular tool to investigate phenotypes and varying environmental conditions. LC-ESI-QTOF-MS/MS analyses of a wide range of metabolites showed that it is an effective method to identify differences and similarities between E. huxleyi strains grown in different conditions. Strain and growth phase appear to be the more important factors in differentiating metabolite profiles. Surprisingly there were no obvious metabolite profiling differences between calcifying and non-calcifying cells. Untargeted analysis can, however, be used to identify the compounds that did display differences, and which may be important biomarkers, so warrant further investigation. A range of metabolite profiling techniques highlighted important differences between strains, which will hopefully lead onto further research into the metabolome of E. huxleyi, and the unravelling of important metabolic pathways. There has been little research into the LMW metabolites of E. huxleyi, and especially comparisons between strains. Thus the use of metabolomics is a novel way to investigate the difference between cell types and the possible functions of calcification.

Changing climate in combination with growing world populations mean that there is growing need for plants to be grown on land that is currently considered marginal for agriculture. Sorghum is a C4 plant that serves as an important food crop in Africa and India. It is also known to be highly drought tolerant but the mechanisms responsible for this tolerance are unclear. The overall aim of this study was to understand the drought tolerance mechanisms that enable the plant to maintain leaf function for a long time during water deficit. In Chapter 2 of this thesis, I studied the underlying physiological mechanisms for tolerating drought in two sorghum varieties with differing degrees of drought tolerance compared to a closely related species, Zea mays. During progressive drought, the more tolerant sorghum variety Samsorg 17 maintained net CO2 assimilation and photochemistry longest relative to the less tolerant Samsorg 40 and Zea mays. Differences were also seen in stomatal aperture, stomatal density, total chlorophyll content, chl a:b and A/Ci curve responses with maize more affected than the sorghum varieties. In Chapter 3, I identified novel drought tolerance mechanisms in the sorghum varieties. The less tolerant Samsorg 40 lost PsbA (D1) and Rubisco proteins and reengineered its photosynthetic apparatus to accumulate amino acids and sugars in order to maximise survival under drought. Samsorg 17 maintained photosynthetic proteins notably PsbA (D1) and Rubisco and accumulated high constitutive sugar content allowing for the maintenance of transpiration and photosynthesis. The two sorghum varieties had strikingly contrasting approaches of tolerating drought as demonstrated in Chapter 3. In Chapter 4, the aim was to characterise biochemical and metabolic changes that occur in response to drought. In particular, to identify sugars that are accumulated constitutively in Samsorg 17 and nitrogen sinks for lost N in Samsorg 40. My findings indicated a contrasting response in terms of sugar content in Samsorg 17 but support for amino acids as N sinks in Samsorg 40 as reported earlier. Sugars, sugar alcohols, lipids, organic acids, heat shock proteins and dehydrins were generally higher or more induced in Samsorg 17 relative to Samsorg 40. Samsorg 40 rather made amino acids. The implications of my findings and future work arising from this study were discussed in detail in the final chapter. In conclusion, in this thesis, it was demonstrated that closely related plants can have mechanistically different physiological and biochemical mechanisms for responding to drought.

Understanding the response of soil microbial communities to various environmental stresses is of current interest, because of their pivotal role in nutrient cycling, soil organic matter mineralization and influence on plant growth. Determining the affect of several biotic and abiotic factors on soil microbial communities is the overall objective of the study. The specific goals are to determine 1) the response of microbial communities to water deficit in soil and 2) how the presence of a rich biotic community determines the direction of microbial community development in cultures. Both goals are novel and unique contributions to understanding microbial ecology in soil. Dynamics in water potentials due to drying and rewetting of soil impose significant physiological challenges to soil microorganisms. To cope with these fluctuations, many microorganisms alter the chemistry and concentration of their cytoplasmic contents. The aim of this research is to understand how the microbial biomass and their cytoplasm change in response to water potential deficits under in situ soil conditions. To address this objective we characterized intracellular and extracellular metabolites in moist, dry and salt stressed soils. Our results provided the first direct evidence that microbial communities in soil in situ utilize sugars and sugar alcohols to cope with low water potential. While the cultivation and isolation of microorganisms is essential to completely explore their physiology and ecology, 99% of soil microbes resist growing in cultures. Presence of very unnatural conditions in the culture plates was considered as main reason for low cultivability. Thus, a culture-based study was conducted whereby microorganisms were grown in association with their native habitat with an objective of mimicking native conditions to promote the growth of previously uncultivated microorganisms. Moreover, the importance of biotic communities (microbe-microbe) and abiotic soil effects were assessed on bacterial growth. Our results strongly indicate that the presence of living microbial community in the vicinity of the target culture resulted in the cultivation of novel members of rare bacterial taxa from phyla Verrucomicrobia, Bacteroidetes, Proteobacteria, and Planctomycetes. These results emphasize the need to develop new culturing methods to tap the hidden microbial potential for emerging anthropogenic needs.

Made available in DSpace on 2014-06-11T19:33:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-28Bitstream added on 2014-06-13T21:06:43Z : No. of bitstreams: 1 carlin_sd_dr_jabo.pdf: 560339 bytes, checksum: 8b2e1669f83b62325f0519f3baf373d4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No presente trabalho foi verificado o ajuste osmótico no crescimento inicial de cana-de-açúcar, cv. IAC91-5155, sob efeito da interação dos estresses hídrico e ácido no solo. Para tanto, foram quantificados os teores de três solutos compatíveis, prolina, glicina betaína e trealose. Também foi estudada a atividade da nitrato redutase e os teores de clorofilas. O estudo de todas estas variáveis teve por objetivo propor a utilização destes compostos como indicadores fisiológicos de aclimatação da cana-deaçúcar sob efeito da interação de ambos os estresses. O experimento foi realizado em casa de vegetação com 29,7±4,3oC e 75±10 UR%. Foram utilizados três tratamentos de disponibilidade hídrica (capacidade de campo, CC): controle (70%), estresse moderado (55%) e estresse severo (40%) e três tratamentos de acidez no solo (saturação por bases, V%): controle (55%), média acidez (33%) e alta acidez (23%). O delineamento experimental utilizado foi o de blocos casualizados em esquema fatorial 3x3, com quatro repetições. Após 60 dias sob estresses foram determinados os teores dos solutos compatíveis, a atividade da nitrato redutase e os teores das clorofilas. Os resultados mostram que houve acúmulo dos três solutos compatíveis evidenciando que ocorreu ajuste osmótico. Prolina e glicina betaína são indicadores fisiológicos do efeito da interação de ambos os estresses enquanto trealose é indicadora apenas para o estresse hídrico. Também, a atividade da nitrato redutase pode ser utilizada como indicador do estresse hídrico e os teores das clorofilas a, b e total para os estresses hídrico ou ácido. A massa de matéria seca da parte aérea, o número de folhas e a área foliar das plantas jovens de cana-de-açúcar, ajustadas osmoticamente, são reduzidos pelos estresses. Os efeitos interativos de ambos... / In this present work was verified the osmotic adjustment in the initial plant growth sugarcane cv. IAC91-5155 under interaction effect of water and acid stress in the soil. For this, were quantified three compatible solutes content, as follows: proline, glycine betaine and trehalose. It was verified also the nitrate reductase activity as well the chlorophyll content. The study of all parameters aimed to propose the use components as physiological indicators to acclimation of sugarcane plants under interaction the both stress. The experiment was carried out in a greenhouse under 29.7±4.3oC and 75±10%. Three water availability treatments (Field Capacity – FC%): control (70%), moderate stress (55%), and extreme stress (40%) and three soil acidification treatment (Base Saturation – BS%): control (55%), average acidity (33%), and high acidity (23%) were used. The experimental design used was the randomized blocks in a 3x3 factorial scheme and four replicates. After sixty days under stress were determined compatible solutes, nitrate reductase and chlorophyll content. Our results showed that there was the accumulation of three compatible solutes exhibiting osmotic adjustment. Glycine betaine and proline are both physiologic indicators of stresses interaction effect whereas trehalose indicates only water stress. The nitrate reductase activity can be used as water stress indicator and chlorophylls a, b and total are indicators to water or acid stress. The dry mass of shoot part, leaves number and leaf area are significantly reduced by stress in young sugarcane plants adjusted osmotically. The interaction of stresses cause decrease of dry mass while water stress reduces leaves number and leaf area.

Orientadora: Durvalina Maria Mathias dos Santos / Banca: Raffaella Rossetto / Banca: Marcelo de Almeida Silva / Banca: Marcos Omir Marques / Banca: David Ariovaldo Banzatto / Resumo: No presente trabalho foi verificado o ajuste osmótico no crescimento inicial de cana-de-açúcar, cv. IAC91-5155, sob efeito da interação dos estresses hídrico e ácido no solo. Para tanto, foram quantificados os teores de três solutos compatíveis, prolina, glicina betaína e trealose. Também foi estudada a atividade da nitrato redutase e os teores de clorofilas. O estudo de todas estas variáveis teve por objetivo propor a utilização destes compostos como indicadores fisiológicos de aclimatação da cana-deaçúcar sob efeito da interação de ambos os estresses. O experimento foi realizado em casa de vegetação com 29,7±4,3oC e 75±10 UR%. Foram utilizados três tratamentos de disponibilidade hídrica (capacidade de campo, CC): controle (70%), estresse moderado (55%) e estresse severo (40%) e três tratamentos de acidez no solo (saturação por bases, V%): controle (55%), média acidez (33%) e alta acidez (23%). O delineamento experimental utilizado foi o de blocos casualizados em esquema fatorial 3x3, com quatro repetições. Após 60 dias sob estresses foram determinados os teores dos solutos compatíveis, a atividade da nitrato redutase e os teores das clorofilas. Os resultados mostram que houve acúmulo dos três solutos compatíveis evidenciando que ocorreu ajuste osmótico. Prolina e glicina betaína são indicadores fisiológicos do efeito da interação de ambos os estresses enquanto trealose é indicadora apenas para o estresse hídrico. Também, a atividade da nitrato redutase pode ser utilizada como indicador do estresse hídrico e os teores das clorofilas a, b e total para os estresses hídrico ou ácido. A massa de matéria seca da parte aérea, o número de folhas e a área foliar das plantas jovens de cana-de-açúcar, ajustadas osmoticamente, são reduzidos pelos estresses. Os efeitos interativos de ambos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this present work was verified the osmotic adjustment in the initial plant growth sugarcane cv. IAC91-5155 under interaction effect of water and acid stress in the soil. For this, were quantified three compatible solutes content, as follows: proline, glycine betaine and trehalose. It was verified also the nitrate reductase activity as well the chlorophyll content. The study of all parameters aimed to propose the use components as physiological indicators to acclimation of sugarcane plants under interaction the both stress. The experiment was carried out in a greenhouse under 29.7±4.3oC and 75±10%. Three water availability treatments (Field Capacity - FC%): control (70%), moderate stress (55%), and extreme stress (40%) and three soil acidification treatment (Base Saturation - BS%): control (55%), average acidity (33%), and high acidity (23%) were used. The experimental design used was the randomized blocks in a 3x3 factorial scheme and four replicates. After sixty days under stress were determined compatible solutes, nitrate reductase and chlorophyll content. Our results showed that there was the accumulation of three compatible solutes exhibiting osmotic adjustment. Glycine betaine and proline are both physiologic indicators of stresses interaction effect whereas trehalose indicates only water stress. The nitrate reductase activity can be used as water stress indicator and chlorophylls a, b and total are indicators to water or acid stress. The dry mass of shoot part, leaves number and leaf area are significantly reduced by stress in young sugarcane plants adjusted osmotically. The interaction of stresses cause decrease of dry mass while water stress reduces leaves number and leaf area. / Doutor

"2003". / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2004. / Bibliography: leaves 136-160. / Introduction: The Antarctic environment; Antarctic inhabitants; Microfungi; Identification of microfungi; Physiological factors affecting Antactic microfungi; Flow cytometry and microfungi; Hydrolytic enzymes of industrial interest; Isolation of genes from microfungi; Aims of this study -- Materials and methods: Fungal strains and cultivation conditions; Molecular identification of fungal isolates; Fungal physiology; Hydrolase activity of secreted proteins; Gene cloning and expression -- Results and discussion: Microfungal identification; Physiological factors affecting Antarctic microfungi; Activity in microfungi when grown on solid media; Characterisation of hemicellulases from selected Antarctic microfungi; Cloning of an Antarctic Penicillium allii lipase gene and its expression in Trichoderma reesei -- Conclusions and future prospects. / The Antarctic occupies that region of the planet that falls below the 60th parallel of South latitude. Although it has been frequented by adventurers, journeyman scientists and tourists for the past 100 years, the Continent has remained virtually unoccupied. The intense cold, the absence of human occupation and the limited range of local higher animal species have combined to create the impression that the Continent is virtually devoid of life. -- Although the microbiota of the Antarctic has attracted some small level of attention in the past, the examination of filamentous microfungi has been largely overlooked and fallen to a small group of dedicated investigators. In this study it will be shown that far from being an insignificant component of the Antarctic network, microfungi represent a potentially large and so far untapped bioresource. -- From just 11 bryophyte samples collected at four sites in the Ross Sea/Dry Valleys region of Southern Antarctica, some 30 microfungal isolates were recovered. Using molecular techniques, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) was sequenced to reveal no less than nine unique microfungal species. For only two of these species did the ITS sequence data produce a 100% match with records held on the public databases. This investigation also highlighted the problems inherent in the traditional morphological identification system which are now being perpetuated in the molecular database records. -- A set of seven notionally identified isolates obtained from ornithogenic soil samples gathered in the Windmill Islands in Eastern Antarctica (offshore from the Australian Antarctic Division's Casey Station) were also subjected to molecular identification based on ITS sequence data. Each of the seven isolates was identified as a unique species; six were cosmopolitan in nature and the one remaining bore very little resemblance at the molecular level to any of the recorded species although it was provided with an epithet commonly used in the identification of Antarctic microfungal species. -- To evaluate their potential as a bioresource, samples of Antarctic microfungi were examined to determine if the same physiological factors common to mesophilic species also applied to their Antarctic analogues. It is known that when placed under stress, trehalose can act as a protectant against cold (cryoprotection) and dehydration in mesophilic yeasts and fungi. The level of trehalose produced by the Antarctic isolates and their mesophilic analogues when subjected to stress was compared. A similar comparison was made for the production of glycerol which is well established as a compatible solute providing protection to mesophilic species against osmotic stress. Only in the case of trehalose production by an Antarctic Embellisia was there any indication that either of these two compounds could play a significant role in providing protection to the Antarctic fungi against the rigours of their environment, which leaves open to question what in fact does. -- In the course of investigating the means by which Antarctic microfungi guard against the damage which can ensue when subjected to oxidative stress, flow cytometry was introduced as an investigatory tool. It was established that there is a window of opportunity during which flow cytometry can be used to undertake a detailed analysis of the early stages of fungal growth from germination through hyphal development. -- Of major significance in determining the potential of Antarctic microfungi as a resource is their ability to produce new and novel enzymes and proteins. The microfungal isolates were screened for hydrolytic activity on solid media containing indicative substrates and proved to be a fruitful source of enzymes active over a range of temperatures. A detailed characterisation of two hemicellulases, β-mannanase and xylanase, secreted into a liquid medium by a subset of the Antarctic fungi and a high producing mesophilic reference strain permitted direct comparisons to be made. It was shown that the maximum hemicellulase activity of the Antarctic strains occurred at least 10°C and as much as 30°C lower than that of the reference strain and that mannanase activity for two of the Antarctic isolates exceeded 40% of their maximum at 0°C. These assay results highlight the potential of Antarctic microfungi to yield novel cold-active enzymes. -- As a final measure of the capacity of the Antarctic to yield novel enzymes from its microfungal stock, a lipase gene was selected as a target for isolation and expression in a heterologous fungal host. Using PCR techniques, the gene of interest was isolated from an Antarctic isolate of Penicillium allii, transformed into the mesophilic production host Trichoderma reesei and the active protein successfully produced in the growth medium. The recombinant lipase was assayed and found to exhibit novel characteristics consistent with a cold-adapted enzyme. / Mode of access: World Wide Web. / 186 leaves ill

La maîtrise du développement des moisissures retrouvées dans le contexte agro-alimentaire parmi les flores microbiennes d'altération ou technologiques répond à des enjeux économiques et sanitaires importants. Le développement des moisissures peut être affecté par des facteurs abiotiques comme la température, l'activité de l'eau (aw) ou la composition gazeuse. L'évaluation de l'effet de ces facteurs via des outils de mycologie prévisionnelle vise à prévoir l'altération fongique des aliments. Ce travail a pour objectif d'explorer l'impact des conditions environnementales sur la physiologie de moisissures d'intérêt pour l'industrie agro-alimentaire.L'effet de la température, de l'aw ajustée par du glycérol ou du chlorure de sodium (NaCl) du pH et de la composition gazeuse a été évalué sur la germination des spores et/ou la croissance radiale de cinq moisissures: Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum et Penicillium roqueforti. Au niveau appliqué, ces travaux ont montré que l'effet du NaCl ou de la composition gazeuse peuvent être inclus dans une approche de mycologie prévisionnelle. Le choix des souches représentatives d'une espèce fongique et l'état physiologique des spores utilisées comme inoculum ont un impact significatif sur les modèles prédictifs.Au niveau fondamental, des marqueurs ont été recherchés pour évaluer l'effet des facteurs abiotiques sur la physiologie des spores. La température et l'aw ont un effet significatif sur l'état physiologique des spores et leur germination. La recherche de marqueurs moléculaire, contribuera aux connaissances de l'effet des facteurs abiotiques sur la physiologie des moisissures. / In the food processing industry, controlling the development of filamentous fungi encountered as spoilers or technological cultures address significant economic and sanitary issues. Fungal development in foods is mainly determined by abiotic factors including temperature, water activity (aw) or the headspace gas composition. The quantification of these respective effects through a predictive mycology approach aims at preventing fungal food spoilage. The present work aims at exploring the effect of environmental conditions on the physiology of filamentous fungi of interest in the food processing industry.The effect of temperature, aw (adjusted with glycerol of sodium chloride), pH and headspace gas composition was evaluated on conidial germination and/or radial growth of five fungi isolated from dairy products: Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum and Penicillium roqueforti. The present work suggests that the specific effects of sodium chloride or gas composition could be included in predictive mycology approaches. It was also demonstrated that the selection of strains representative of a fungal species and the physiological state of conidia utilized as inoculum have a significant effect on the final predictive models.At the fundamental level, markers were investigated to study the effect of abitoic factors on the physiological state of spores. The temperature and aw significantly affected the physiological state of spores and their germination kinetics. The investigation of markers at the molecular level could provide better knowledges on the effect of abiotic factors on the physiology of filamentous fungi.

Living cells accumulate compensatory solutes for protection against the harmful effects of extreme environmental conditions such as high salinity, temperature and desiccation. Even at high concentrations these solutes do not disrupt the normal cellular functions and at times counteract by stabilizing the cellular components. These properties of compensatory solutes have been exploited for stabilizing proteins and cells in vitro. Betaines are widespread natural compensatory solutes that have also been used in other applications such as therapeutic agents and polymerase chain reaction (PCR) enhancers. Some biomedical applications of novel synthetic analogues of natural betaines were investigated. Natural compensatory solutes are either dipolar zwitterionic compounds or polyhydroxyl compounds, and the physical basis of compensation may differ between these, so one focus was on synthetic betaines with hydroxyl substituents. The majority of the synthetic solutes stabilized different model proteins against stress factors such as high and low temperatures. The presence of hydroxyl groups improved protection against desiccation. The observed stabilization effect is not just on the catalytic activity of the enzyme, but also on its structural conformation. Synthetic compensatory solutes have a potential application as protein stabilizers. Dimethylthetin was evaluated as a therapeutic agent and found to be harmful in a sheep model. However, from the study we were able to generate a large-animal continuous ambulatory peritoneal dialysis (CAPD) model and showed that glycine betaine could be added to the dialysis fluid in chronic renal failure. Some synthetic compensatory solutes reduce the melting temperatures of DNA better than most natural solutes. Synthetic solutes were identified that have potential to enhance PCR and could replace some reagents marketed by commercial suppliers. Density, viscosity and molecular model data on the solutes showed correlations with the biochemical effects of the solutes, but no physical measurements were found that reliably predicted their potential for biotechnological applications.