Most plants complete their life cycle in a single location and therefore are affected by the changing environment. As a result, plants have evolved physiological and developmental adaptations to overcome stress. The work presented in this thesis has examined the regulation of photosynthetic electron transport in barley, rice and Thellungiella salsuginea. Barley is considered as a crop which is comparatively tolerant to soil salinity. The focus of this study was to evaluate the physiological responses of photosynthesis in barley under salinity and to characterize traits responsible for the regulation of photosynthesis. At low salt concentrations, barley plants protect PSII centres from excitation pressure by down-regulating the electron transport chain and maintaining ΔpH, by cyclic electron transport associated with PSI, to support nonphotochemical quenching (NPQ). However, at the highest concentration of salt examined, this regulation starts to fail. The failure might result from a specific loss of PSI, resulting in reduced cyclic electron flow, or an increase in the leakiness of the thylakoid membranes, resulting in loss of ΔpH. The effects of salinity on the regulation of electron transport through Photosystem I and Photosystem II have been studied in two rice varieties from Sri Lanka. The regulation of photosynthesis in the salt-tolerant At-354 is more prominent than in the salt-sensitive Bg-352 when plants are exposed to salt. Exposure of Bg-352 to salt resulted in a substantial decrease in gas exchange, PSII photochemistry, leaf area and loss of chlorophylls. The decrease in the photosynthesis in AT-354 is caused by stomatal limitations, which restrict the CO2 entry into the plants, whereas the decrease of photosynthesis in Bg-352 is caused by non-stomatal limitations. Results suggest that At-354 protects PSII centres from excitation pressure by down-regulating the electron transport chain and maintaining ΔpH by cyclic electron transport associated with PSI to support NPQ. At high salt concentration, this regulation starts to fail in Bg-352.Tolerance to abiotic and biotic stress has evolved in many wild plant species, termed extremophiles. These plants contain essential genes which may used to improve crop production in changing environments. Thellungiella salsuginea is an extremophile, able to grow and reproduce in extreme environments. Stepien and Johnson (2009) identified a protein, known as the plastid terminal oxidase (PTOX) which acts as an alternative electron sink in T. salsuginea under salt stress. The current study showed that, in addition to salt, T. salsuginea showed increases in PTOX protein content and activity when exposed to drought, different growth irradiances and cold with high light. Semi-natural conditions also triggered the activity of PTOX. This study also showed that salt caused an up-regulation of PTOX gene transcripts in the leaves of salt treated T. salsuginea plants compared to control plants. Direct electron transport from PSII to PTOX and then to oxygen via the PQ pool accounted for up to 30% of total PSII electron flow in T. salsuginea (Stepien and Johnson, 2009). Efficient electron flow from PSII to PTOX would however, probably require co-location of these complexes in the same thylakoid fraction. To examine the location of PTOX in the thylakoid membrane, immunoblot analyses were performed, to test for changes in other protein complexes which may be associated with PTOX. In addition blue-native polyacrylamide gel electrophoresis and immunoblots were performed to isolate and detect the PTOX protein with any associated complexes. Although immunoblot analysis showed a prominent signal, mass spectrometry data did not allow identification of PTOX. This results suggests that further studies are needed to identify the precise localisation of the PTOX protein in the thylakoid membranes in T. salsuginea.

Les contraintes environnementales dans un monde en changement remettent en cause la survie des plantes ; les plus drastiques sont les contraintes abiotiques, comme les stress hydriques, thermiques, ou la pollution par les métaux lourds. Les mécanismes inductibles de la tolérance des plantes sous stress ont été étudiés par une approche physiologique combinée à une analyse protéomique. Des plants de Populus tremula x P. alba genotype 717-1B4 ont été exposés à plusieurs contraintes en chambre phytotronique : 1) un sol contenant des concentrations importantes de Cd2+ ou Zn2+, 2) une sécheresse induite par arrêt d'arrosage, 3) une contrainte thermique par élévation subite ou graduelle de 22 à 42°C. Les paramètres physiologiques des plantes ont été suivis au cours des traitements ainsi que durant la période de recouvrement qui a suivi les contraintes hydriques et thermiques. La réponse de stress a été caractérisée par ces paramètres physiologiques et par les changements dans les profils protéomiques du tissu foliaire et de la zone cambiale. Les aspects communs et particuliers du stress induit par chaque contrainte ont été décrits. Les données rassemblées dans cette étude, en enrichissant les connaissances sur la gamme de réponse des essences ligneuses, contibuent à déterminer la frontière entre le stress générique et les réponses plus spécifiques ; elles apportent également des éléments de réponse à l'utilisation du peuplier en phytoremédiation des sols pollués par les métaux. / Environmental constraints in a changing world challenge plants to survival. Among these constraints, the most drastic are the abiotic ones, e.g. water stresses or temperature stresses and dispersaI of inorganic pollutants like heavy metals. The inducible tolerance mechanisms of the stressed plant were investigated by a physiological approach coupled with an in-depth proteomic study. Populus tremula x P. alha genotype 717IB4 cuttings were exposed to diverse constraining environments in phytotrons: 1) soil added with Cd2+ or Zn2+, 2) drought induced by withholding water, 3) heat constraint, either through heat shock or stepwise increased temperature from 22°C to 42°C. Physiological parameters of the plants were monitored throughout the exposure to constraint and, for water and heat stresses, recovery period as well. Plant stress responses were characterized by physiological parameters and changes in proteomic patterns in leaf and cambial tissues. Common and specifie features of the stress induced by each of the constraints were described. The data gathered during this study enrich the knowledge on trees range response capabilities, contribute to shape the borders between generic and specifie stress response and open potential use of poplar in phytoremediation of metal polluted sites.

Soybean (Glycine max) and rice (Oryza sativa) are the most important crops cultivated worldwide. The productivity of both crops is severely limited due to drought stresses. Abscisic acid (ABA) signaling is one of the crucial phytohormones which acts as the signaling mediator in different environmental stress for adaptive response of plants. In this study, functional characterization of abscisic acid-activated protein kinase-like kinase 1 (AALK1), and low molecular weight protein tyrosine phosphatase (LMWPTP) were studied by developing gain-ofunction and loss-ofunction phenotypes by transgenesis. Physiological response of AALK1 showed that AALK1 modulates the drought stress response ins soybean plants. The study has demonstrated several key genes are differentially expressed control, and aalk1-RNAi silenced lines under drought treatment. The AALK1 overexpression lines enhanced the transcription of other ABA-responsive genes, indicating that the AALK1 is a positive regulator of ABA-mediated stress signaling pathways in soybean. The phylogenetic analysis and domain analysis also supports that AALK1 is abscisic acid-activated protein kinase and has a role in drought response. Phenotype analysis of LMWPTP in rice showed that transgenic overexpression of LMW-PTP exhibited significantly improved drought tolerance in comparison to RNAi silencing and control plants ,which indicates that LMWPTP modulates the drought stress tolerance of rice plants. Further, 5 putative tyrosine phosphorylated proteins were detected through immunoblotting and identified by mass spectrometry. Some of these tyrosine phosphorylated proteins are likely to be target proteins of LMWPTP. Together, the present findings strengthen the knowledge about the functional role of AALK1 and LMWPTP, which can be utilized as a promising gene-based molecular marker in transgenic breeding for generating crop plants with improved drought tolerance which ultimately improve the grain yields.

Many environmental stress factors have been identified that increase square and boll abscission and thus result in reduced cotton (Gossypium hirsutum L.) yield. Under stress conditions, ethylene, an endogenous hormone, is elicited. Ethylene peaks before abscission to promote the formation of the abscission layer and plays a major role in early season square and boll abortion. In addition, ethylene stimulates the leaf senescence process. Thus, it is desirable to protect a crop from ethylene-induced fruit loss and premature leaf senescence under stress conditions. The overall objective of this study was to test the hypothesis that the ethylene inhibiting compound 1-methylcyclopropene (1-MCP) treatment can have a beneficial effect on the physiology, biochemistry and yield traits of cotton plants under abiotic stress conditions under field and controlled environment studies. The growth chamber studies were conducted in 2011 using a randomized complete design with six replications. Cotton plants were exposed to stress conditions (heat and drought) and 1-MCP treatment at the seven-true-leaf stage. The heat stress study consisted of two 1-MCP rates (0 and 10 g a.i. ha-1) and two temperature regimes (optimum temperature: 30/20 ºC (day/night temperature) and high temperature: 40/25 ºC). The drought study consisted of two water regimes (well-watered and water-stressed) was exposed to two rates of 1-MCP (0 and 10 g a.i. ha-1). Both of the field studies were conducted with a randomized complete block design with four replications in 2010 and 2011 at the Texas A&M AgriLIFE Research Farm in Burleson County, TX. The objective of the first field study was to evaluate the ability of 1-MCP to protect cotton plants against abiotic stress imposed by a foliar treatment of ethephon. Eight treatments consisted of two 1-MCP rates (0 and 10 g a.i. ha-1) in combination of four ethephon rates (0, 146, 292, and 438 mL ha-1) were imposed at the first flower (FF) stage of crop development. The second field study investigated the effect of 1-MCP on boll development and the corresponding subtending leaves, and consisted of two 1-MCP rates (0 and 10 g a.i. ha-1) applied at 20 days after flowering. In the growth chamber study examining heat and drought, application of 1-MCP resulted in reductions of lipid peroxidation, membrane leakage, and soluble sugar content as well as increased chlorophyll content, compared to the untreated plants under stress conditions. In the field study to evaluate the effect of 1-MCP under ethephon stress, 1-MCP increased plant height and number of main stem nodes in both years. In addition, 1-MCP treated plants exhibited greater membrane integrity and increased photosystem II quantum efficiency, and thus delayed senescence in both years. This potential for yield increase was realized in 2011 with 1-MCP treatment exhibiting a higher lint yield. In 2012, although 1-MCP treatment increased number of open fruit and open fruit weight per plant, no significant yield increase was detected. In the field study to test the effect of 1-MCP on boll development and subtending leaf conditions, 1-MCP treatment increased cotton boll weight at 20 days after flowering. One probable explanation for the enhanced boll size was the healthier subtending leaves: 1-MCP-treated subtending leaves exhibited decreased membrane damage and lipid peroxidation, and higher chlorophyll content and photosynthetic efficiency.

Philosophiae Doctor - PhD / Sorghum bicolor (L.) Moench is an important cereal crop currently explored as a potential bio-energy crop due to its stress tolerance and ability to ferment soluble sugars. Physiological studies on sorghum varieties have demonstrated that part of drought tolerance is attributed to sugar accumulation in the sorghum stems. Despite the agronomic advantages of sorghum as a bio-energy crop, more research efforts towards the molecular elucidation of sorghum traits that confer drought tolerance are necessary. Particular focus on traits, which could potentially contribute to an efficient bio-energy production under environmental constraints, would be an added advantage. This study examined the role of sugar biosynthesis proteins in conferring tolerance to drought-induced hyperosmotic stress, and ultimately osmotic adjustment in sorghum varieties. Sorghum bicolor (L.) Moench varieties (ICSB338, ICSB73, ICSV213 and S35) with different levels of drought tolerance, were grown under watered conditions until early anthesis after which, a 10-day water deficit period was introduced

Doctor of Philosophy / Department of Horticulture and Natural Resources / Sunghun Park / Drought and heat stress are two of the biggest constraints to global food production. Abiotic stress response pathways are complex and consist of osmotic adjustors, macromolecule stabilizers, and antioxidants to counteract the damaging nature of abiotic stress induced reactive oxygen species (ROS) accumulation. In this work, we studied the effect of overexpression of an Arabidopsis glutaredoxin, AtGRXS17, on heat tolerance in maize (Zea mays L.) and drought tolerance in rice (Orzya sativa L.). Glutaredoxins (GRXs) are proteins cable of reducing disulfide bonds, therefore regulating the cellular redox status, and require glutathione for regeneration. Ectopic expression of AtGRXS17 in maize resulted in increased heat stress tolerance during flowering. AtGRXS17 enhanced heat tolerance by increasing kernel set and total grain yield during heat treatments, compared to wild type controls. Our results indicated that AtGRXS17-expressing maize plants produce heat tolerant pollen with higher germination rates than wild type when challenged during heat treatments. Furthermore, AtGRXS17-expressing plants were less susceptible to post pollination heat induced kernel abortion. Rice plants expressing AtGRXS17 were also tolerant to abiotic stress. AtGRXS17-expressing rice was more tolerant to drought stress challenges and consistently survived drought treatments. A nontargeted metabolomics study revealed distinct changes in profiles of key metabolite groups in response to drought stress. Soluble sugars and amino acids accumulate as osmotic adjustors while antioxidants, such as glutathione, accumulate to mediate ROS accumulation and regulate redox activity. All genotypes accumulated amino acids, soluble sugars, and raffinose family oligosaccharides in response to drought stress. Our results indicated AtGRXS17-expression affected several pathways known to increase drought tolerance. Altered sugar metabolites suggested a redox modulation of sucrose synthase activity and significant increases in the secondary sulfur assimilation pathway metabolites suggested altered sulfur metabolism. This research provides new insights into ability of GRXs to improve heat tolerance and crop yield in maize and functions of GRXs in affecting metabolite profiles contributing to increased drought tolerance in rice.

>Magister Scientiae - MSc / South Africa is one of two countries responsible for the production of approximately 80% of the world’s Zr. The increase in mining activity has detrimental effects on the environment, especially crop plants, as more pollutants are leached into the soil. Consequently, it is necessary to understand how plants respond to this form of abiotic stress. Therefore, this study focused on determining the physiological and biochemical responses of two genotypes of Brassica napus L (Agamax and Garnet) in response to Zr stress. The levels of cell death, lipid peroxidation and ROS were higher in Garnet, whereas the chlorophyll content was higher in Agamax. Furthermore, native PAGE analysis detected seven SOD isoforms and seven APX isoforms in Agamax, compared to 6 SOD isoforms and 7 APX isoforms in Garnet. The results thus indicate that Agamax is tolerant to Zr-induced stress, whereas Garnet is sensitive. An assay for the rapid quantification of Zr within plant samples was subsequently developed, which revealed that Agamax retained the bulk of the Zr within its roots, whereas Garnet translocated most of the Zr to its leaves. The ability of Agamax to sequester Zr in its roots comes forth as one of the mechanisms which confers greater tolerance to Zr-induced stress. As a consequence, our study sought to use the optical, physical and chemical properties of quantum dots to image the uptake and translocation of Zr in B. napus genotypes. ICPOES was also performed to quantify Zr levels in various plant organs. Data from the ICPOES revealed varying patterns of uptake and translocations between Garnet and Agamax. These patterns were similarly shown in IVIS Lumina images, tracing the transport of QD/Zr conjugates. This method ultimately proved to be successful in tracing the uptake of Zr, and could essentially be a useful tool for targeting and imaging a number of other molecules.

The Asteraceae family is frequently used to describe several medicinal plants which contain various phytochemical compounds including phenols, flavonoids and terpenoids. Among the Asteraceae family German chamomile (Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) plants are extant species used in contemporary medicine. These phytochemical compounds have been traditionally used since ancient times in health care systems worldwide as a source of medicines. The use of micropropagation is essential to improve and increase these active compounds via plant tissue culture within a short period of time using the application of key plant growth regulators (PGRs). Furthermore, quantitative and qualitative analysis using high performance liquid chromatography- ultraviolet detector (HPLC-UV) and gas chromatography- flame ionisation detector (GC-FID) of potential medicinal compounds expressed by both chamomile and yarrow are important points. The protocol of in vitro shoots, roots and callus formation of chamomile and yarrow seeds culture were investigated using Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs). MS culture medium containing 0.5 mgL-1 IAA and 1.0 mgL-1 of GA3 were found to be the best culture medium for chamomile and yarrow seeds. In this project in vitro and in vivo growth rates of selected plant species were also investigated. In the earlier growth stages yarrow plants were found to grow much quicker than chamomile, while the yield of chamomile flowers was significantly (p ≤ 0.001) more than yarrow flowers. The phenolic, flavonoid and terpenoid compounds content of leaves and flowers of plants produced from both cultures were also studied. HPLC-UV analysis showed that chlorogenic acid, apigenin-7-O-glucoside and luteolin dominated as the main phenol and flavonoid compounds recovered in both in vitro and in vivo chamomile and yarrow cultures. However, GC-FID analysis indicated that farnesene and nerolidol were detected as the main terpenoid compounds present in the two culture conditions used to grow chamomile and yarrow plants. Moreover, this research examines how chamomile and yarrow plants can produce and improve their phytochemical compounds content not only under well-watered conditions but also under drought stress conditions. The main phenol and flavonoid compounds of chlorogenic acid, caffeic acid, apig-7-glucoside, umbelliferon and luteolin were found in chamomile and yarrow varieties grown under both well-watered and drought stress conditions using (HPLC-UV), however farnesene, nerolidol, chamazulene, α-(-)- bisabol and bisabolol oxide A were observed in the plant essential oils (EOs) using Soxhlet extraction and GC-FID analysis. The antibacterial activity of plant EOs was also investigated using disc diffusion and 96 well plates. In vivo chamomile EO showed the highest antibacterial activity against gram-positive and gram-negative bacteria strains. In addition, in vitro yarrow EO showed the greatest effect on the death of bacteria strains.

N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some root development parameters somewhat more tolerant than WT plants, but more sensitive in terms of shoot growth. Collectively the data suggests that FAAH activity may interact with stress-responsive pathways in plants, perhaps including pathways involving ABA.

In the United Kingdom and Ireland, a major percentage of fertilized agricultural area is devoted to grasslands, which helps to support the associated milk and beef production industries. In temperate grasslands, perennial ryegrass (L. perenne) is the major forage grass and this species is particularly suitable as a forage grass due to its high yield and digestibility, when compared with other species. However, perennial ryegrass is not well adapted to abiotic stress conditions which are likely to occur in its natural environment. Some of the abiotic stress factors which have significant impacts on plant growth and development include water and nutrient availability. Therefore, this project set out to unravel some of the mechanisms involved in the adaptation of perennial ryegrass to limited water, phosphorous and nitrogen. In order to understand the metabolic mechanisms acting in response to these stresses, metabolite profiling was performed using GC-MS. Furthermore, for the water- and phosphorous-limitation studies this approach was complemented with transcript analysis.In order to study water-limitation a hydroponics system supplemented with polyethyleneglycol (PEG) was used to induce water-limitation for a period of one-week. A clear difference in the metabolic profiles of the leaves of plants grown under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more water stress-susceptible genotype Cashel and an increase in sugars and compatible solutes in the drought-tolerant PI 462336 genotype. Sugars exhibiting a significant increase included, raffinose, trehalose, glucose, fructose and maltose. Raffinose was identified as the metabolite exhibiting the largest accumulation under water-stress in the more tolerant genotype and may represent a target for engineering superior drought tolerance or form the basis of marker-assisted breeding in perennial ryegrass. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336 which identified genes in perennial ryegrass that were regulated by this stress.The characterization of the response to phosphorus-limitation was performed in a hydroponics system containing two solutions with different levels of phosphorus. Samples were collected from the roots and leaves of two genotypes 24 hours after being exposed to stress. Internal phosphate concentrations were reduced and significant alterations were detected in the metabolome and transcriptome of two perennial ryegrass genotypes. Results indicated a replacement of phospholipids with sulfolipids in response to P deficiency and that this occurs at the very early stages of P deficiency in perennial ryegrass. Additionally, the results suggested the role of glycolytic bypasses and the re-allocation of carbohydrates in response to P deficiency The characterization of the metabolic response of L. perenne leaves to different levels of nitrogen supply was performed for seven different genotypes with variability in the regrowth response rate to nitrogen supply in a hydroponics system. This facilitated the identification of common mechanisms of response between genotypes to nitrogen. The metabolic response observed included modifications of the lipid metabolism, as well as alterations of secondary aromatic metabolite precursors in plants exposed to nitrogendeficit. In contrast, plants grown in a nitrogen saturated media appeared to modify to some extent the metabolism of ascorbate. Additionally, it was found that amino acid levels increased with increasing concentrations of nitrogen supplied. This study suggested that the involvement of secondary metabolism, together with lipid and ascorbate metabolism, is of crucial importance in the early-adaptation of perennial ryegrass plants to different levels of nitrogen supply.