Young barley seedlings (Hordeum vulgare L.) were stressed using nutrient solutions containing NaCl or polyethyleneglycol (PEG) and measurements were made of leaf growth, water status, proline soluble sugar contents of growing (basal) and non-growing (blade) tissues. Leaf growth ceased within seconds following exposure of seedlings to osmotic solutions with water potential values (ψ) = -3 to -11 bars but growth resumed after a lag period. Latent periods were increased and new growth rates were decreased as ψ of nutrient solutions were lowered. Growth ceased before detectable changes occurred in tissue water status but leaf basal tissues began to adjust osmotically, and reductions of 1 to 2 bars in both ψ and osmotic potential (π) usually occurred for the first 1 to 2 hours with lower reduction rates thereafter. After 1 to 3 days exposure of seedlings to solutions with different ψ, cumulative leaf elongation was reduced as the ψ of the root medium was lowered. Reductions in ψ and π of tissues in leaf basal regions paralleled growth reductions, but turgor (P) was largely unaffected by stress. In contrast, ψ, π and P of leaf blades were usually changed little regardless of the degree and duration of stress, and blade ψ were always higher than ψ of basally located cells. It is hypothesized that blades have high ψ and are generally unresponsive to stress because water in most of the mesophyll cells in this area does not exchange readily with water present in the transpiration stream. Measurements of proline contents in different sections of leaf following water stress, showed that in living tissues proline levels are dynamically related to water status of the tissue. In the basal regions where reductions in ψ and π occurred rapidly, proline levels were elevated quickly, whereas, accumulation of proline in mid-blade tissues occurred slowly and in lower concentrations. The combined data of many experiments showed a strong correlation between proline levels and tissue ψ (r = 0.93) and π (r = 0.85). Increase in total soluble sugars (TSS) and ion concentrations, contributed significantly to the stress-induced osmotic adjustment observed in the growing tissue.

Jiang, Tiantian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 61-75). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.v / Acknowledgements --- p.vi / List of Figures and Tables --- p.vii / Chapter Part 1 --- Introduction --- p.-0- / Chapter 1.1 --- Brassinosteriods (BRs) and BR signaling --- p.-0- / Chapter 1.1.1 --- Discovery of BRs --- p.-2- / Chapter 1.1.2 --- Functions of BRs --- p.-4- / Chapter 1.1.3 --- BR signaling pathway --- p.-6- / Chapter 1.2 --- Nitrogen (N) and N responses --- p.-10- / Chapter 1.2.1 --- Hormones involved in plant N responses --- p.-11- / Chapter 1.3 --- Anthocyanin and anthocyanin synthesis --- p.-13- / Chapter 1.3.1 --- Anthocyanin structures --- p.-13- / Chapter 1.3.2 --- Functions of anthocyanins --- p.-14- / Chapter 1.3.3 --- Biosynthesis of anthocyanins --- p.-14- / Chapter 1.3.4 --- Regulations of anthocyanin biosynthesis --- p.-15- / Chapter 1.4 --- Hormones and plant nutrient stresses --- p.-19- / Chapter Part 2 --- Materials and Methods --- p.-20- / Chapter 2.1 --- Plant materials and growth conditions --- p.-20- / Chapter 2.2 --- Measurement of anthocyanin content --- p.-21- / Chapter 2.3 --- Yeast two-hybrid (Y2H) assay --- p.-22- / Chapter 2.4 --- Bimolecular fluorescence complementation (BiFC) assays --- p.-23- / Chapter 2.5 --- Quantitative real-time PCR --- p.-25- / Chapter 2.6 --- Electrophoretic mobility shift assay (EMSA) and competition assay --- p.-26- / Chapter 2.7 --- Histochemical staining of GUS activity --- p.-28- / Chapter Part 3 --- Results --- p.-29- / Chapter 3.1 --- 24-epibrassinolide (24-eBR) increases plant tolerance to N-starvation in Arabidopsis - --- p.-29- / Chapter 3.2 --- BR treatment enhances anthocyanin accumulation under N deprivation conditions --- p.-31- / Chapter 3.3 --- BZR1 interacts with PAP1 in vitro and in vivo --- p.-35- / Chapter 3.4 --- BR and BZR1 promote the expression of the 'late' anthocyanin biosynthetic genes during N deprivation - --- p.-39- / Chapter 3.5 --- BZR1 binds to the promoter of DFR --- p.-43- / Chapter 3.6 --- BR-enhanced anthocyanin accumulation is specific to N-deprivation --- p.-46- / Chapter 3.7 --- BZR1 differently regulates PAP1 and PAP2 --- p.-48- / Chapter 3.8 --- Endogenous GL3 is required for BR-enhanced anthocyanin biosynthesis --- p.-52- / Chapter 3.9 --- N status affects the expression of BR biosynthetic gene CPD --- p.-52- / Chapter Part 4 --- Discussion --- p.-54- / Chapter 4.1 --- BRs confer plant tolerance to low-N stress and the tolerance is mediated by BR enhancement of low-N-induced anthocyanin biosynthesis --- p.-54- / Chapter 4.2 --- BRs enhance anthocyanin accumulation under N starvation through BZR1-PAP1 interaction or direct control of the expression of anthocyanin biosynthetic genes --- p.-55- / Chapter 4.3 --- BRs are specifically involved in low-N induced anthocyanin production --- p.-56- / Chapter 4.4 --- Transcription factors that specifically control BR-regulated anthocyanin biosynthesis --- p.-57- / Chapter 4.5 --- DFR is an important target of BR-regulation of anthocyanin biosynthesis --- p.-58- / Chapter Part 5: --- Conculsions --- p.-59- / Chapter Part 6: --- References --- p.-61-

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.

Graduation date: 1991

Protocols for the successful micropropagation of 'Queen Elizabeth' ('Q.E.') and 'Dick Koster' ('D.K.') roses were established, yielding a seven-ten fold multiplication rate per month. The effects of mechanically induced stress (MIS) (shaking stress) were evaluated on early establishment of greenhouse-grown 'Q.E.' and 'D.K.' rose cuttings and the ex vitro survival and hardiness of micropropagated 'Q.E.' plantlets. Shaking 'Q.E.' rose cuttings at 200 rpm for 30 min daily for 4 weeks during the rooting stage increased root length, dry weight and the root:shoot dry weight ratio. Similar shaking of 'D.K.' rose at 200 rpm for 15 min increased shoot fresh and dry weight and root length and dry weight. Prior to ex vitro acclimatization, plantlets shaken at 150 rpm for 15 min had reduced leaf dry weights. Those shaken at 200 rpm for 15 min had lower specific root water content but greater percent root dry matter. MIS was not directly implicated in improving ex vitro survival and hardiness of 'Q.E.' rose. (Abstract shortened by UMI.)

No description available.

No description available.