abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Plants regulate many physiological processes in response to adverse environmental stresses. This study focused on the seed germination and seedling establishment stage and investigated the molecular signaling events when abiotic stresses, such as osmotic, water and temperature, were applied. Seeds of Arabidopsis, mutants or wild type, were used to identify the signaling components. Cold-pretreatment (stratification) is widely used to break seed dormancy and improve germination rate. Stratification at 4. significantly broke the seed dormancy of Arabidopsis in wild-type, cyp707a2, sleepy1 and sleepy1/cyp707a2, but not in ga3ox1. Stratification and exogenous ABA treatment strongly enhanced the expression and the activity of a-amylase in the freshly harvested seeds among the wild-type and those mutants, which have relatively high ABA content. Similarly, the expression of RGL2 and ABI5 were also substantially suppressed by stratification. These results suggest that stratification firstly leads to GA biosynthesis and unlocks the inhibition of RGL2 on the expression of a-amylase. Stratification also relieves the inhibition of ABA on the germination process but the inhibition of ABA on seedling development is not affected. We have isolated an Arabidopsis mutant, dsptp1, which is hyposensitive to osmotic stress during seed germination and seedling establishment, indicated by exhibiting higher seed germination rate, lower inhibition in root elongation under osmotic stress, and more tolerance to drought compared with the wild type (Col0) plants. Osmotic stress and drought enhanced AtDsPTP1 expression in seed coats, the bases of rosette leaves and roots. Compared with the wild type, the dsptp1 mutant increased proline accumulation, reduced MDA content and ion leakage, and enhanced antioxidant enzyme activity under osmotic stress. AtDsPTP1 regulated the transcript levels of various dehydration responsive genes, ABA biosynthesis and metabolic enzyme gene under osmotic stress, resulting in reduced accumulation of ABA in dsptp1 mutant plants than wild type in response to osmotic stress. AtDsPTP1 also mediated the ABA signaling pathway under osmotic stress by suppressing the expression of ABI1 and enhancing the expression of the positive regulators ABI3 and ABI5 in ABA signaling. These data suggest that AtDsPTP1 positively regulates ABA accumulation and signaling during seed germination and seedling establishment in Arabidopsis under osmotic stress. To further investigate the regulation mechanism of DsPTP1 in osmotic stress and drought signaling, we analyzed the water holding capacity between wild type and dsptp1 mutant. The dsptp1 mutant exhibited enhanced water holding capacity compared to wild type under osmotic stress resulting from reduced water loss and increased relative water content, which shall contribute the osmotic and drought tolerance. To identify the signaling components, we investigated the activity of MAPKs under osmotic and drought stress and found that the DsPTP1 differentially regulates the activities of MAPK6 and a p38 MAPK, which is inferred as MAPK12 according to its molecular weight in Arabidopsis under osmotic and salt stress. However, there is no direct interaction between DsPTP1 and 20 MAPKs indicated by the results of the of specific interaction test. These results suggest that the differential regulation of MAPK6 and MAPK12 by DsPTP1 is indirect. In addition, we screened the interaction proteins of DsPTP1 under abiotic stress. Seventeen positive clones were acquired from the sequencing results. More work need to be done to confirmed the positive interactions and the signaling cascades. In summary, seed germination and seedling growth are closely regulated by environmental cues. This should be the result of evolutionary selection since successful new growth from the seed embryo depends on the sensitive perception of environmental conditions and effective regulation of many physiological processes that are involved. We have demonstrated that plant hormones, especially ABA, play central regulative roles during such regulations. Many other signaling components, such as protein kinases and phosphatases, are also involved. Identifying the detailed signaling pathways should be the focus of further research.

The alga Dunaliella salina possesses the unique ability to accumulate up to 14 % of it's dry weight as β-carotene in response to stress conditions. This hyper-accumulation of β-carotene has led to the commercial exploitation of this alga for the biotechnological production of this important carotenoid. In order to maximise β-carotene production, a dual-stage process which separates the distinctive growth phase from the β-carotene accumulating stress phase has recently been patented. Preliminary laboratory studies showed that although stress factors such as high salinity and nutrient limitation enhance β-carotene accumulation in D. salina (± 10 pg.cell⁻¹), high light intensity was the single most important factor contributing to the induction of β-carotene accumulation in this alga (± 20 pg.cell⁻¹). Moreover, it was demonstrated that β-carotene accumulation can be further stimulated by exposing the alga to a combination of high light intensity, salt and nutrient stresses (± 30-60 pg.cell⁻¹). The response of D. salina to stress was shown to occur in two phases. The first phase occurred within 24 hours and was characterized most importantly by higher rates of β-carotene accumulation for all the stresses investigated. In cells exposed to multiple stress factors in mass culture, the β-carotene accumulation rate was as much as 9.5 pg.cell⁻¹.day⁻¹ in the first phase compared to only 3 pg.cell·day⁻¹ in the second phase. Since the rate of β-carotene accumulation was higher within the first 24 hours after exposure to stress, the first phase was considered crucial for stress-induced β-carotene accumulation. Characterization of this phase revealed that the stress response was multifaceted. Transition of cells from the growth stage to stress conditions was characterized by the following: 1) Change in cell volume. Hypersalinity caused cell shrinkage while cells exposed to nutrient limitation and/or high light intensity caused cells to swell. Restoration of cell volume was shown to occur within 8 hours for all stresses investigated. 2) Altered photosynthesis. Inhibition of both carbon fixation and oxygen evolution was demonstrated in cells immediately after exposure to multiple stress factors. 3) Production of abscisic acid. Intracellular ABA levels increased within 6-8 hours after exposure to all stresses investigated. The rise in intracellular ABA levels coincided with an increase or return to starting cell volume. High intracellular ABA levels were however transient and within 24 hours ABA began to partition into the culture medium. 4) Change in pigment composition. Changes in xanthophyll cycle pigment content was demonstrated soon after exposure to stress conditions. In hypersalinity shocked cells, initial epoxidation of zeaxanthin to violaxanthin and subsequent de-epoxidation to zeaxanthin occurred, whereas exposure to high stress resulted in an immediate and continued decrease in the epoxidation state indicating accumulation of zeaxanthin. A rapid rate of chlorophyll depletion was also demonstrated. In addition to the above responses a rapid decrease in growth rate during this phase was also noted. An interrelationship between cell volume change, ABA production, maintenance of xanthophyll cycle operation and β-carotene accumulation therefore appeared to exist. ABA production was shown to be stoichiometrically related to changes in xanthophyll content with r² = 0.84 and slope of the curve = 0.91 being achieved for high light stressed cells. This study therefore presents strong circumstantial evidence in support of a carotenoid origin for ABA in Dunaliella. In addition, enhanced β-carotene content was achieved by the application of exogenous ABA and related compounds suggesting a role for ABA as a regulator of the overall stress response. Furthermore, zeaxanthin accumulation was shown to be positively correlated ( r²≥ 0.81) to β-carotene accumulation for all the stresses investigated. The second phase was characterized by a return to homoeostasis of the physiological processes mentioned above, indicating acclimation of the cell to prevailing conditions. This stage was characterised by continued β-carotene accumulation and a decreased epoxidation state of the xanthophyll cycle which together appeared to sustain photosynthesis, allowing this organism to tolerate stress conditions.

The alga Dunaliella salina accumulates large quantities of β-carotene in response to certain environmental and physiological stresses. This hyper-accumulation process has been commercially exploited. However, the currently employed averaging or single-stage process produces β-carotene yields well below the genetic potential of the organism due to the inverse relationship between growth and secondary metabolite production. A dual-stage process, which separates the distinctive growth and secondary metabolite production stages of the alga, has been proposed. The broad aim of the research programme was to evaluate the practicality, scale-up and economic viability of a dual-stage β-carotene production process from D. salina. Preliminary laboratory studies showed that although stress factors such as high salinity and a range of nutrient limitations enhance β-carotene accumulation in D. salina, high light intensity is the single most important factor inducing β-carotene hyper-accumulation in the alga. Furthermore, the preliminary studies indicated that 6-carotene production could be successfully manipulated by the imposition of stress. The stress response of D. salina to high light stress was examined at a fundamental level. The relative partitioning of β-carotene between thylakoid membrane and interthylakoid globular β-carotene has revealed two responses to high light stress. The first is a response in which the alga adapts to the photoinhibitory effects of high light stress by the rapid accumulation and the peripheral localisation of Jl-carotene to the outer extremities of the chloroplast. This is followed by a maintenance response which is characterised by the recovery of the photosynthetic rate and cell growth. A possible interrelationship between the extent of the photo inhibitory response and the amount of β-carotene hyper-accumulation has been noted. An outdoor evaluation of the growth stage of the dual-stage system has demonstrated that D. salina can be grown in a relatively low salinity, nutrient sufficient medium for extended periods without overgrowth by small non-carotenogenic Dunaliella species. In addition, biomass productivities of three times greater than those obtained in the currently employed averaging system were achieved. The role of high light intensity in β-carotene hyper-accumulation was confirmed in outdoor scale-up stress pond studies. The studies demonstrated the feasibility of stress induced ll-carotene production in outdoor cultures of D. salina and β-carotene yields three times greater than those obtained in the currently employed averaging process were achieved. The dual-stage process imposes the specific requirement of viable cell separation on the harvesting system employed. A flocculation-flotation process and an air-displacement crossflow ultrafiltration system were developed and successfully evaluated for the separation of D. salina from the brine solution in a viable form. The extraction of β-carotene from D. salina was evaluated. Supercritical fluid extraction studies showed that the use of a co-solvent mixture of carbon dioxide and propane could effectively reduce the high extraction pressures associated with supercritical carbon dioxide extraction. In addition, a novel hydrophobic membrane assisted hot oil extraction process was developed which separates the complex oil-water emulsions produced during hot oil extraction of 6-carotene from wet D. salina biomass. Process design and economic evaluation studies were undertaken and showed that the economics of the dual-stage process offer significant advantages over the currently employed averaging process.

No description available.

Leaves of young barley (Hordeum vulgare cv Arivat) seedlings were examined anatomically, physiologically and cytologically to infer the pathway of transpirational water movement and to understand the basis for the selective responsiveness of the growing region to osmotic stress. Vessels with open lumens were found to extend from the intercalary meristem to the expanded blade, and all vessels are present in 5 functional vascular bundles (FVB) which are separated by 20 to 30 closely packed mesophyll cells and 2 to 3 immature vascular bundles (IVB). Heat pulse transport data confirmed the anatomical suggestion that water will move throughout the leaf in open vessels and they showed also that osmotic stress will reduce water transport within 1 min, which is before transpiration is lowered. Water representing about 2 per cent of the total tissue water was obtained by centrifuging cut sections of the growing region at 5 X g against an adsorptive surface. This water is probably xylem plus cell wall water because it is easily removed, its volume is 2X that calculated to be in the vessels, and it exchanges more readily with the water in the nutrient solution than the bulk tissue water. This lack of free exchange indicates apoplastic water is somewhat separated from mesophyll cells, and it is hypothesized that osmotic stress causes sudden growth cessation and initation of metabolic changes because (a) reduced water availability together with ongoing transpiration will cause a sudden reduction in the xylem's water potential, (b) there is a lateral transmission of this reduced water potential through walls of all cells in the growing region, and (c) cells can respond in some way to changes in water potential around them. Most cells in the expanded blade are considered unresponsive to osmotic stress because transpirational water will move predominantly from the 5 FVB through the closest stomata, so only cells closest to those bundles will be altered rapidly by stress.

Significant alterations in the environmental conditions can have pronounced effects on plant genome stability. Recent evidence argues for a global involvement of the components of epigenetic modules in the regulation of genome homeostasis both immediately after stress exposure and long after environmental cues were acquired. The last observation is of particular interest as the memory of imposing stress can be maintained at the molecular level throughout plant ontogenesis and may be faithfully propagated into the following generation. Our study provides evidence that epigenetic repercussions exerted by stress exposure of parental plants manifest themselves in untreated progeny at all three levels of the epigenetic module: DNA methylation, histone posttranslational modifications and small RNA metabolism. Additionally, the results of our study shed new light on the engagement of the epigenetic machinery in the maintenance of plant genome integrity by counteracting the activity of invading nucleic acids. / xv, 280 leaves : ill. ; 29 cm

by Li Wing Yen Francisca. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 94-105). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgemnets --- p.vii / Abbreviations --- p.ix / Table of contents --- p.xii / List of figures --- p.xvi / List of tables --- p.xvii / Chapter 1. --- General Introduction / Chapter 1.1 --- Introduction to oxidative stress / Chapter 1.1.1 --- Introduction to Reactive Oxygen Species --- p.1 / Chapter 1.1.2 --- Major sites of ROS production / Chapter 1.1.2.1 --- Chloroplast --- p.4 / Chapter 1.1.2.2 --- Mitochondria --- p.4 / Chapter 1.2 --- Regulation of intercellular ROS content in plant cells / Chapter 1.2.1 --- Enzymatic defense ofROS --- p.6 / Chapter 1.2.1.1 --- Superoxide dismutases --- p.6 / Chapter 1.2.1.2 --- "Ascorbate peroxidase, Glutathione reductase and the Ascorbate-Glutathione cycle" --- p.7 / Chapter 1.2.1.3 --- Catalase --- p.11 / Chapter 1.2.1.4 --- Alternative oxidase --- p.11 / Chapter 1.2.2 --- Non-enzymatic / Chapter 1.2.2.1 --- Ascorbate and Glutathione --- p.12 / Chapter 1.2.2.2 --- α-tocopherol --- p.12 / Chapter 1.3 --- "Salt, dehydration and oxidative stress" / Chapter 1.3.1 --- Oxidative stress is induced when plants were under salt stress --- p.13 / Chapter 1.3.2 --- Oxidative stress is induced when plants were under dehydration stress --- p.14 / Chapter 1.4 --- ROS scavenging: the road to achieve multiple-stress tolerance? --- p.16 / Chapter 1.5 --- Purple acid phosphatase and its relationship with oxidative stress in plants / Chapter 1.5.1 --- General introduction to plants purple acid phosphatase (PAP) --- p.20 / Chapter 1.5.2 --- Purple acid phosphatases that found to be involved in ROS scavenging in plants --- p.21 / Chapter 1.6 --- Previous studies in GmPAP3 --- p.23 / Chapter 1.7 --- Hypothesis and significance of this project --- p.25 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- "Plants, bacterial strains and vectors." --- p.26 / Chapter 2.1.2 --- Chemicals and reagents --- p.27 / Chapter 2.1.3 --- Commercial kits --- p.28 / Chapter 2.1.4 --- Primers and adaptors --- p.29 / Chapter 2.1.5 --- Equipments and facilities used --- p.31 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.31 / Chapter 2.1.7 --- Software --- p.31 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Molecular techniques / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.32 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.32 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5α, DE3 and Agrobacterium competent cells" --- p.33 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.36 / Chapter 2.2.1.5 --- DNA and RNA extraction --- p.37 / Chapter 2.2.1.6 --- Generation of single-stranded DIG-labeled PCR probes --- p.38 / Chapter 2.2.1.7 --- Testing the concentration of DIG-labeled probes --- p.40 / Chapter 2.2.1.8 --- Northern blot analysis --- p.40 / Chapter 2.2.1.9 --- PCR techniques --- p.41 / Chapter 2.2.1.10 --- Sequencing --- p.42 / Chapter 2.2.2 --- Plant cell culture and transformation / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.43 / Chapter 2.2.2.2 --- Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells --- p.44 / Chapter 2.2.3 --- Growth and treatment conditions for plants / Chapter 2.2.3.1 --- Growth and salt treatment condition of soybean samples for gene expression studies of GmPAPS --- p.45 / Chapter 2.2.3.2 --- Root assay of GmPAP3 transgenic Arabidopsis thaliana --- p.46 / Chapter 2.2.4 --- "Immunolabeling, mitochondria integrity, ROS detection and confocal microscopy" / Chapter 2.2.4.1 --- Immunolabeling of GmPAP3-T7 transgenic cell lines --- p.47 / Chapter 2.2.4.2 --- Mitochondria integrity --- p.48 / Chapter 2.2.4.3 --- Detection of Reactive oxygen species (ROS) --- p.48 / Chapter 2.2.4.4 --- Confocal microscopy --- p.49 / Chapter 2.2.4.5 --- Images processing and analysis --- p.49 / Chapter 2.2.5 --- Statistical analysis --- p.50 / Chapter 3. --- Results / Chapter 3.1 --- "Expression of GmPAP3 was induced by NaCl stress, oxidative stress, and dehydration stress" --- p.51 / Chapter 3.2 --- Establishment of GmPAP3-T7 fusion transgenic cell lines / Chapter 3.2.1 --- Subcloning of GmPAP3-T7 into the binary vector system W104 --- p.53 / Chapter 3.2.2 --- Transformation of W104-GmPAP3-T7 into tobacco BY-2 cells --- p.56 / Chapter 3.3 --- Establishment of GmPAP3 trangenic cell lines / Chapter 3.3.1 --- Subcloning of GmPAP3 into the binary vector system W104 --- p.58 / Chapter 3.3.2 --- Transformation of W104-GmPAP3 into tobacco BY-2 cells --- p.58 / Chapter 3.4 --- Establishment of GmPAP3 transgenic Arabidopsis thaliana / Chapter 3.4.1 --- Transformation of W104-GmPAP3 into Arabidopsis thaliana --- p.61 / Chapter 3.5 --- Colocalization of GmPAP3 with MitoTracker-orange --- p.66 / Chapter 3.6 --- Effect of expressing GmPAP 3 on mitochondria integrity of BY-2 cells under NaCl and dehydration stress. --- p.71 / Chapter 3.7 --- Effect of expressing GmPAP3 on ROS production in BY-2 cells under salt and PEG treatment --- p.75 / Chapter 3.8 --- Effect of expressing GmPAP3 in Arabidopsis thaliana under salt stress --- p.81 / Chapter 4. --- Discussion / Chapter 4.1 --- Gene expression profile of GmPAP3 --- p.83 / Chapter 4.2 --- Subcellular localization of GmPAP3 --- p.84 / Chapter 4.3 --- Functional tests of GmPAP 3 transgenic BY-2 cells / Chapter 4.3.1 --- GmPAP3 could protect the plant cells' mitochondria integrity when under salt and dehydration stress --- p.86 / Chapter 4.3.2 --- Expressing GmPAPS in tobacco BY-2 cells were able to reduce the production ofROS under salt and dehydration stresses --- p.88 / Chapter 4.4 --- Functional tests of GmPAP3 transgenic Arabidopsis --- p.91 / Chapter 5. --- Conclusion and perspectives --- p.92 / References --- p.94 / Appendix I: Restriction and modifying enzymes --- p.106 / Appendix II: Chemicals --- p.107 / Appendix III: Commercial kits --- p.111 / Appendix IV: Equipments and facilities used --- p.112 / "Appendix V: Buffer, solution, gel and medium formulation" --- p.113

Pacific lampreys (Lampetra tridentata) have declined in abundance in the Columbia River Basin. Although, the reasons for the decline are unclear, we suggest that development of hydroelectric dams and habitat alterations in tributaries as the main causes. The available knowledge of life history of Pacific lampreys and status from dam counts (trend data) in the Columbia River Basin and the Umpqua River along the Oregon Coast shows that populations have been declining over the last 30 years. Even though Pacific lampreys have been shown to have ecological importance both as predator and prey, the declines in their populations have been largely ignored by fisheries agencies and the public. Recently, the National Marine Fisheries Service initiated studies on using radio-telemetry of Pacific lampreys in order to study the impact of hydroelectric dams on migration behavior. To address one of the fundamental assumptions of radio-telemetry, namely, that tagged fish are "normal," one must be able to measure whether or not an animal is stressed. We identified clinical indicators of stress in adult Pacific lampreys. Plasma glucose became elevated soon after acute stress and remained elevated for one week. Plasma lactate also became elevated by 30 minutes; however, it decreased to resting levels by one hour after stessor. Muscle lactate was shown to have an inverse relationship with glucose. Muscle lactate levels decreased by 4 hours and remained depressed for two days. Plasma chloride ions decreased by one hour, then returned to resting levels by 8 hours; by 24 hours, levels were again decreased with recovery occurring by 48 hours. The steroid cortisol was not found in the plasma of Pacific lampreys. The swimming performance and physiological effects of surgical implantation of three different sized dummy radio transmitters in Pacific lampreys were assessed. Intraperitoneal implantations of 3.4 g transmitters had no significant effect on circulating levels of glucose (an indicator of stress) 4 months after surgery, while 10 gram transmitters showed a significant increase in plasma glucose. Lampreys implanted with 7.4 g transmitters recovered from surgery by day 4 based on levels of plasma glucose. Lampreys implanted intraperitoneally with 7.4 g dummy transmitters showed no significant differences in circulating glucose 30, 60, 90, and 180 days after surgery in comparison to sham-implant controls. Ventilation rate decreased significantly by 30 minutes after surgery and was stable by 60 minutes; suggesting initial recovery from surgery is rapid. Swimming performance was impaired immediately after surgery; however, swimming was not compromised at 1 and 7 days after surgery. Tagged fish showed a significant difference in oxygen consumption when tested immediately after surgery; however, oxygen consumption was at control levels at 1 and 7 days after surgery. / Graduation date: 2001

Organismal tolerance to abiotic environmental stresses contributes significantly to setting the distribution limits of organisms, as demonstrated by vertical zonation patterns in the marine intertidal zone. In this thesis, the ultimate (evolutionary) and proximate (mechanistic) causes of tolerance to temperature and emersion stresses associated with the intertidal zone were examined using porcelain crabs, genus Petrolisthes. Species of Petrolisthes from intertidal and subtidal microhabitats of four biogeographic regions of the Eastern Pacific were used in phylogenetically-based comparative analyses of morphological, physiological, and biochemical adaptation to environmental stress. A phylogenetic tree based on the sequence of the 16sRNA gene was developed to facilitate these analyses. Organismal thermal tolerance limits are adapted to match maximal microhabitat temperatures. Acclimation of thermal tolerance limits suggests that temperate intertidal zone species are living close to their thermal maximum in nature. Respiratory responses to emersion vary among species from different vertical zones. Experimental examination of oxygen consumption rates and lactate accumulation during emersion suggests that intertidal species are able to respire in air using thin membranous regions on the ventral meral segments of their legs (leg membranes). Leg membrane size is positively correlated with body size across species, but not within a single species. Evolutionary analyses indicate that leg membranes may not have evolved for purposes of aerial respiration, but their presence may have allowed intertidal and subtidal species to achieve larger body sizes and higher metabolic rates. The thermal stabilities of an enzyme, lactate dehydrogenase (LDH), from 22 species of Petrolisthes varied widely, but were not correlated with maximal habitat temperatures. Comparative analyses did not indicate any evolutionary relationship between LDH thermal stability and microhabitat conditions. Experimental evidence suggests that interspecific differences in LDH stability are genetically based, and are due both to intrinsic properties of the LDH molecules and extrinsic protein stabilizers. Elucidation of the mechanism(s) of LDH stabilization in Petrolisthes may provide novel insight to the field of protein stabilization. These results studies suggest that individual traits may be subjected to differing levels of selection, and thus the analysis of environmental adaptation requires careful consideration of the biological significance of the traits being examined. / Graduation date: 1999