Large, membrane-bound vacuoles of plant cells are suitable organelles for the compartmentation of ions. These vacuoles contain Na+/H+ antiporters for movement of Na+ within the organelle in exchange for H+. They provide an efficient mechanism to prevent the occurance of detrimental outcomes of Na+ accumulation in the cytosol. Identification of AtNHX1 gene that confers resistance to salinity by expressing a Na+/H+ antiport pump facilitates the understanding of the salt stress tolerance mechanisms of plants. The aim of the present study was to isolate and clone the Arabidopsis thaliana AtNHX1 coding sequence for transformation of Nicotiana tabacum plants via Agrobacterium tumefaciens mediated gene transfer. For this purpose, total RNA was isolated from Arabidopsis thaliana plants and cDNA synthesis was performed. AtNHX1 (1614bp) was amplified by using cDNA of Arabidopsis via specific primers. The amplified PCR product was verified by sequencing. AtNHX1 coding sequence was cloned into the plant transformation vector pCVB1 and 10 independent putative transgenic tobacco plants were obtained via Agrobacterium tumefaciens mediated gene transfer sysytem. Transfer of selected 8 putative transgenic plants to soil provided the regeneration of T1 seeds. Germination of the seeds under different salt treatments (0, 50, 100, 150, 200, 250 mM NaCl) was observed for evaluating the salt tolerance of transformed plants. The 82% and 60% of the transgenic T1 seeds were germinated on 150 mM NaCl and 200 mM NaCl containing media, respectively. In contrast the germination percentage of wild type tobacco seeds under 150 mM NaCl and 200 mM NaCl concentrations were 39% and 21%, respectively. The germination rate of the transgenic T1 seeds were significantly higher (p=0,001) when compared to the control seeds especially under high salt stress conditions (150 and 200 mM NaCl). Taken all together, our results demonstrated that the germination efficiencies and growth of the plants transformed with AtNHX1 were higher than the wild type tobacco plants under high salt concentrations.

Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 16 p.

Thèse doctorat : Sciences du Vivant. Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 20 p.

Thèse doctorat : Sciences du Vivant. Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. Thèse doctorat : Sciences du Vivant. Aspects Moléculaires et Cellulaires de la Biologie : Université de Fudan - Shanghai - Chine : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 10 p.

Soil application of a known activator of Systemic Acquired Resistance (SAR), benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH), and Harmonizer, a polychlorinated copper (II) phthalocyanine pigment, reduced severity of Colletotrichum orbiculare in Nicotiana benthamiana by 99% and 38%, respectively. BTH induced expression of nine SAR/progammed cell death-related genes and primed expression of two Induced Systemic Resistance (ISR)-related genes, while Harmonizer induced expression of only one SAR-related gene. Soil application of Harmonizer also reduced severity of Sclerotinia homoeocarpa in Agrostis stolonifera up to 39%, whereas BTH was ineffective. Next generation sequencing identified over 1000 genes in A. stolonifera with two-fold or higher increased expression following Harmonizer treatment relative to a water control, and induced expression of three defense-related genes was confirmed by relative RT-PCR. These results demonstrate that Harmonizer can activate systemic resistance in a dicot and a monocot, but changes in expression of genes indicated that it differed from BTH-activated SAR. / Petro-Canada, Natural Sciences and Engineering Research Council of Canada, Ontario Turfgrass Research Foundation

Life as we know it would be impossible in the absence of oxygen. However, too much oxygen can be toxic to the aerobic organisms which depend on it for their very existence. This apparent paradox arises as a result of oxygen's ability to accept electrons, forming highly reactive (reduced) oxygen species such as superoxide, hydrogen peroxide and the hydroxyl radical. The toxicity of oxygen is greatly enhanced in illuminated plants, due to the photosynthetic reactions which produce both oxygen and highly energetic electrons in close proximity to one another. These problems are further exacerbated when plants are exposed to a variety of stress conditions, since these conditions reduce the ability of plants to utilise excess electrons. As a result of the danger posed by· these reactive oxygen species, plants have· evolved a complex antioxidant system for their scavenging. Research has shown that plants with naturally elevated levels of the components of the antioxidant system are better equipped to deal with stress conditions which enhance the production of reactive oxygen species. A considerable amount of research has thus been dedicated to the elucidation of the antioxidant system. Almost as much research has been dedicated to enhancing the antioxidant system, with the aim of improving plant productivity under stress conditions. This study sought to evaluate plants carrying elevated levels of two of the enzymes of the antioxidant system. For these purposes, tobacco was transformed with the gene for E. coli glutathione reductase (GR), an enzyme believed to catalyse the rate limiting reaction in the scavenging of hydrogen peroxide. This gene was fused to the gene for the RUBISCO small subunit transit peptide - a peptide capable of targeting proteins to the chloroplast. Due to the presence of this peptide the transformed plants exhibited high chloroplastic levels of GR activity. These plants were crossed with a second tobacco transformant carrying high levels of chloroplastic tomato superoxide dismutase (SOD) - an enzyme responsible for the scavenging of superoxide. These hybrid plants were shown to exhibit high GR and SOD activities in the chloroplast .- the subcellular compartment most susceptible to damage caused by reactive oxygen species. The transgenic hybrids were evaluated for their ability to tolerate oxidative stress by treating them with paraquat - a herbicide whose mode of action involves the production of large quantities of activated oxygen. Under stress conditions, plants carrying just E. coli GR showed a slight improvement in their ability to deal with oxidative stress. In contrast to this, the SOD transformants showed more cellular damage than untransformed control plants. This was attributed to the inability of other enzymes in the antioxidant pathway to deal with the increased flow of metabolites through the pathway. The hybrid transformants showed enhanced stress tolerance in the initial stages of oxidative stress, but this declined with ongoing exposure to stress conditions. As with the SOD transformants, this decline in protection was . ascribed to the relatively low activities of the other enzymes in the antioxidant pathway. It was concluded that elevated levels of the two enzymes conferred greater stress tolerance than just one of the enzymes, but for true stress tolerance it will be necessary to evaluate the antioxidant system and enhance the activity of further enzymes in the pathway. It may also be necessary to improve the regulation of transgene expression, ensuring that none of the enzymes are overwhelmed by the increased flow of metabolites through the system. / Thesis (M.Sc.)-University of Natal, 1996

The responses of Nicotiana tabacum L. var. Samsun to alterations in the nitrogen (N) supply under saline conditions in vitro were monitored. The aim was to test the hypothesis that nitrate-nitrogen supplementation to salt stressed plants alleviates the deleterious effects of salt on plant growth. Due to its capacity to be maintained under stringent environmental conditions, in vitro shoot cultures were chosen as the system of study. Nicotiana tabacum plantlets regenerated from callus in vitro were excised and rooted on solid MS culture medium containing a range of concentrations of NaCI (0 - 180 mM) and N (0 - 120 mM, as NO3--N, NH4+-N or a combination). A variety of parameters of root and shoot growth, nutrient utilisation and nitrogen metabolism were assessed over a 35 d period. Plant growth on 40 mM NO3--N + 20 mM NH4+-N (standard MS nutrients) was inhibited by the presence of salt, with root growth being more adversely affected by salt than stem growth. Root emergence was delayed from 6 d (0 mM NaCI) to 15 d (180 mM NaCI). Similar suppression of growth for all parameters, except root mass and leaf chlorophyll content, was observed when NaCI was replaced with mannitol at equivalent osmolalities. Root mass and leaf chlorophyll were significantly improved in plantlets supplied with mannitol. The time of root emergence was unaffected by mannitol supply, with all roots emerging after 10 d in culture. Plantlet growth on NH4+-N only (0 - 60 mM) was severely inhibited, even in the absence of NaCI, and was inferior to growth on NO3--N. Nitrate additions to salt stressed plantlets could not match growth in control (0 mM NaCI) plantlets. When plantlets were cultured on NO3--N only (0 mM, 30 mM, 60 mM, 120 mM), the increase in nitrate supply up to 60 mM resulted in a small improvement in growth on 90 mM NaCI, but had almost no effect on growth at 180 mM NaCl. A nitrate supply of 120 mM led to growth inhibition in all parameters, even in the absence of NaCl. Plantlet growth on isosmotic concentrations of mannitol in the presence of 0 - 120 mM NO3--N essentially mimicked that of NaCI, except for leaf chlorophyll content which was improved on mannitol at all NO3-·N levels. Nitrate uptake (measured as depletion from growth medium) by plantlets grown on 0 - 180 mM NaCI was positively correlated to availability of nitrate but negatively correlated to NaCI supply. Similar results were obtained for a mannitol supply except nitrate uptake was enhanced significantly on mannitol compared to NaCl. Sodium and chloride uptake appeared unaffected by nitrate concentration. Leaf protein content responded favourably to an increase in the NO3--N supply up to 60 mM and, in particular, appeared to be stimulated in the presence of 180 mM NaCl. Nitrate reductase (NR) activity was found to be inhibited drastically by salt and NO3--N supplementation to the salt medium had no effect on enzyme activity. A reduction in leaf total RNA content was recorded with an increase in NaCI concentration from 0 - 180 mM. A positive response to an increase in the NO3--N supply from 30 mM to 60 mM was detected in the presence of NaCl. Attempts were made to assess the levels of mRNA for NR in response to the various NaCl and N regimes. The plasmid pBMCI02010 containing a NR cDNA insert was isolated and purified and used in both radioactive and non-radioactive RNA slot blot hybridisation procedures. However, due to problems of non-specific binding of the probe, no quantification of the levels of NR mRNA in response to the various treatments could be made. Nitrate supplementation to plantlets of Nicotiana tabacum growing in vitro did not appear to ameliorate the effects of salinity stress, such that growth of plantlets in the presence of NaCI was always inferior to that in the absence of NaCl. As a large portion of growth inhibition was found in this study to be a result of osmotic rather than ionic effects of salt, it is questioned whether a nitrate supply would have an ameliorating effect on plant growth under field conditions. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

Salinity is one of the limiting factors of crop development. Together with causing water loss from plant tissues, salinity also leads to ion toxicity. Under salt stress, increase in Ca+2 concentration in cytosol can decrease the deleterious effects of stress. The binding of Ca+2 to calmodulin initiates a signaling cascade involving the activation of certain transcription factors like STO and STZ. This signal transduction pathway regulates transport of proteins that control net Na+ influx across the plasma membrane and compartmentalization into the vacuole. Previously Arabidopsis STO was identified as a repressor of the yeast calcineurin mutation. Genetical and molecular characterization of STO / a putative transcription factor that takes role in salt stress tolerance can provide a better understanding in the mechanism of salt tolerance and development of resistance in higher plants. The aim of the present study was to amplify and clone the Arabidopsis thaliana sto gene in plant transformation vectors and use them for the transformation of Nicotiana tabacum and Arabidopsis thaliana plants via Agrobacterium tumefaciens mediated gene transfer systems. T0 and T1 progeny of transgenic plants carrying sto were analysed for the stable integration of transgenes, segregetion patterns, expression of the gene and their tolerance to salt stress. The results of the study showed that all transgenic Nicotiana tabacum lines are differentially expressing a transcript that is lacking in control plants and most transgenic lines exhibited higher germination percentages and fresh weights, lower MDA contents under salt stress. On the other hand overexpression of sto in Arabidopsis plants did not provide an advantage to transgenic plants under salt stress, however the anti-sense expression of sto caused decreased germination percentages even under normal conditions. According to the sto expression analysis of wild type Arabidopsis plants, sto was shown to be induced under certain stress conditions like cold and sucrose, whereas it remained constant in salt treatment. External application of plant growth regulators had no clear effect on sto expression, with the exception of slight induction of expression with ABA and ethylene treatments.

Ethylene (C2H4) is a very simple molecule, a gas, and has numerous effects on the growth, development and storage life of many fruits, vegetables and ornamental crops. In higher plants, ethylene is produced from L-methionine in essentially all tissues and ACC Synthase and ACC Oxidase are the two key enzymes in the biosynthesis of ethylene. The objective of the present study was to transform tobacco (Nicotiana tabacum L. cv. Samsun) plant with partial sequence of torenia acc oxidase gene in antisense and sense orientations via Agrobacterium-mediated gene transfer system, and to analyze its effect on ethylene production in transgenic plants. Six antisense and seven sense T0 putative transgenic lines were obtained and were further analyzed with several assays. Leaf disc assay and chlorophenol red assay under selection (75 mg/L kanamycin) revealed positive results compared to the non-transformed plant. T1 generations were obtained from all putative transgenic lines. PCR analysis and Northern Blot Hybridization results confirmed the transgenic nature of T1 progeny. Furthermore, ethylene amount produced by flowers were measured with gas chromatography, which resulted in an average of 77% reduction in S7 line and 72% reduction in A1 line compared with the control flowers. These results indicated that, transgenic tobacco plants carrying torenia acc oxidase transgene both in antisense and sense orientations showed reduced ethylene production thus a possibility of flower life extension.

Terpenes are important specialised metabolites produced by all organisms. Plants produce the greatest diversity of terpenoid compounds, which function in a variety of crucial processes including regulation of growth and development, energy production and plant-insect communication, including pollinator attraction and prevention of herbivore damage. Isopentenyl diphosphate (IPP), the building block for all terpenoid compounds, is synthesised in plants via two unique terpene synthesis pathways located in the plastids and the cytosol, and the regulation of these pathways is still not well understood. The aim of this research was to (1) modify and study the regulation of floral volatile production in Nicotiana tabacum (tobacco) by altering the expression of various enzymes in the terpene biosynthesis pathway and (2) determine the role of specific volatile compounds in floral odour blends in feeding and oviposition behaviours of Helicoverpa armigera, a polyphagous moth of widespread agricultural importance. Expression levels of several enzymes in the terpene biosynthetic pathway were altered by genetic modification in order to modify terpene volatile emissions produced by flowers of N. tabacum. Genes chosen for overexpression were cloned from several species and RNAi hairpins were constructed from gene fragments amplified from tobacco flower cDNA. Transgenic plants were produced by Agrobacterium-mediated transformation, and lines with high levels of transgene expression selected for analysis. The flower-specific Antirrhinum majus chalcone synthase promoter was chosen to control gene expression in transgenic lines in order to avoid the potentially deleterious effects of widespread disruption to terpene biosynthesis. Floral volatiles were sampled using two methods; solid phase microextraction, a highly sensitive technique able to detect even trace levels of volatile compounds in headspace samples, and Tenax sampling, a robust and replicable method to quantify volatile emissions. All floral headspace samples were analysed by gas chromatography-mass spectrometry. Floral volatile analysis determined that wild type Ti68 tobacco flowers emit a simple blend of floral volatiles, with only linalool, a monoterpene, and β-caryophyllene, a sesquiterpene, detected by both sampling methods. Volatile emissions were not subject to temporal regulation, but changes in the floral odour blend were detected during flower development. Overexpression of the plastidic terpene biosynthesis genes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranyl diphosphate synthase did not affect volatile production, however increased farnesyl diphosphate synthase expression in the cytosol surprisingly caused an increase in linalool emissions, synthesised in the plastids. Downregulation of DXR resulted in an albino phenotype affecting all young leaves, the upper stem and the sepals in the most severely affected lines. A significant three-fold decrease in floral linalool emissions, and a nine-fold reduction of both linalool and β-caryophyllene retained within floral tissues was observed in the transgenic lines. In feeding behaviour tests, male and virgin female H. armigera moths did not discriminate between wild type and DXR knockdown flowers at close-range, despite the significant difference in linalool emissions. Expression of an (E)-β-ocimene synthase gene controlled by the CHS promoter did not result in any transgenic plants emitting the novel monoterpene, (E)-β-ocimene. Significant problems with seed germination suggested that (E)-β-ocimene may cause embryo lethality in these lines. However, overexpression of a heterologous (S)-linalool synthase under control of the constitutive cauliflower mosaic virus 35S promoter resulted in a significant two-fold increase in volatile linalool, and β-glycosidase assays confirmed sequestration of a glycosylated linalool derivative in floral tissues. Oviposition preference tests with mated female H. armigera moths indicated a significant preference for egg-laying on wild type flowers compared to flowers with increased linalool production. The results of this research, and previous studies of volatile production in transgenic tobacco, indicate that IPP precursor exchange occurs predominantly in one direction from the cytosol to the plastids, at least under the stress caused by alterations in pathway flux. Regulation of the cytosolic terpene biosynthetic pathway upstream of IPP synthesis appears to be less strictly controlled than the plastidic pathway. Insect behavioural assays support the findings of recent studies in other moth species, and suggest that close-range feeding attraction of H. armigera may be more strongly influenced by visual cues, whereas odour cues, including contact chemoreception, play a more important role in oviposition preferences. The increase in knowledge of the olfactory contribution toward insect-plant communication demonstrated here, and from future work, will lead to improved management of pest species in agricultural and ecological settings.