As the third most important greenhouse gas, nitrous oxide (N2O) is a stable greenhouse gas and also plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N2O stems from the use of nitrogen in agriculture, with soils being the major contributors. Currently, the annual N2O emissions from this “soil–microbe-plant” system is more than 2.6 Tg (one Tg equals a million metric tons) of N2O-N globally. My doctoral studies aimed to explore innovative strategies for N2O mitigation, in the context of environmental microbiology’s potential contribution to alleviating global warming. The bacterial enzyme nitrous oxide reductase (N2OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. Therefore, to “scrub” or reduce N2O emissions, bacterial N2OR was heterologously expressed inside the leaves and roots of transgenic plants. Others had previously shown that the functional assembly of the catalytic centres (CuZ) of N2OR is lacking when only nosZ is expressed in other bacterial hosts. There, coexpression of nosZ with nosD, nosF and nosY was found to be necessary for production of the catalytically active holoenzyme. I have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other four nos genes were coexpressed. More than 100 transgenic tobacco lines, expressing nosZ and nosFLZDY under the control of rolD promoter and d35S promoter, have been analyzed by PCR, RT-PCR and Western blot. The activity of N2OR expressed in transgenic plants, analyzed with the methyl viologen-linked enzyme assay, showed detectable N2O reducing activity. The N2O-reducing patterns observed were similar to that of the positive control purified bacterial N2OR. The data indicated that expressing bacterial N2OR heterologously in plants, without the expression of the accessory Nos proteins, could convert N2O into inert N2. This suggests that atmospheric phytoremediation of N2O by plants harbouring N2OR could be invaluable in efforts to reduce emissions from crop production fields.

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.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Includes bibliographical references: leaf 337-367.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--Mississippi State University. Department of Agricultural Economics. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 199-213). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 199-213). Also available on the Internet.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) is an important glycolytic enzyme and catalyses the reversible conversion of fructose-6-phosphate (Fr-6-P) and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fr-1,6-P2) and inorganic phosphate (Pi). Sugarcane PFP has been inversely correlated with sucrose content across segregating F1 varieties. The down-regulation of PFP in cultivar NCo310 in a previous study led to an increase in sucrose accumulation and fibre content in immature tissue. Several potential transgenic sugarcane lines from genotypes 88H0019 and N27, transformed with the untranslatable sense sugarcane PFP-β gene, were characterized in this study. Initial screening for transgenesis was determined by slot blot and Southern blot analysis to confirm the presence of the co-transformed selectable marker npt II transgene. Northern blot analysis confirmed expression of the 1.2 kb PFP-β transcript in 7 of 9 lines analyzed. Sugar analysis using standard South African Sugarcane Research Institute (SASRI) mill room practices and HPLC was performed on 12 month old pot grown stalks divided into immature and mature tissue sections. The analysis of wild type 88H0019 showed an average sucrose content of 17.84 and 30.76 g sucrose/stalk in immature and mature tissue, respectively. However, no significant difference between the putative transgenic plant values and wild type controls was seen. PFP specific activity was determined in these tissues using enzymatic assay analysis and although levels obtained in immature tissue were between 5-18 nmol/min/mg protein, they were less than values previously reported in sugarcane. The results indicated that no down-regulation of PFP in immature tissue occurred when comparing transgenic and wild type plants. A more discrete internodal tissue sampling method was used to overcome the difficulty of detecting small changes in PFP enzyme activity in bulked stalk tissue sections. Fine analysis of PFP was conducted on specific developmental tissues and single stalks were divided into immature (internodes 1-3), maturing (internodes 4-5) and mature (internodes 7-8) regions. Sucrose analysis was performed using HPLC and PFP activity was determined enzymatically on each tissue type. The analysis of discrete developmental tissues showed specific PFP activity of 60-80 nmol/min/mg protein in young tissue, an amount which falls in the range previously obtained for sugarcane. However there was no significant difference between PFP or sucrose in the transgenic lines when compared with the wild type controls in any of the three developmental tissues examined. Western blotting and densitometric analysis of the blots confirmed the lack of PFP down-regulation in immature tissue in all lines. A final analysis of PFP iv in immature stalk tissue on selected lines was performed using quantitative PCR, which became available near the end of the study. The fold change of each transgenic line indicated that there was a minor increase in PFP confirming the lack of effect of transgenesis. Although evidence for the expression of the PFP-β transgene was seen in the northern blot, no further evidence for transgenesis could be found to support the desired effect of down-regulation of PFP. Characterization of transgenic stalks in this study was hindered by a limited number of lines available for analysis and large variability between replicate samples. Sampling techniques employed in an attempt to make use of existing standard SASRI mill room practices for sugar analysis highlighted the need for a more precise sampling method, specifically when determining the effects of an enzyme manipulation such as PFP. A refined approach has been developed which will assist researchers in the choice of analytical techniques for screening and characterization of potential transgenic lines in the future.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / Fructose 2,6-bisphosphate (Fru 2,6-P2) is an important regulatory molecule in plant carbohydrate metabolism. There were three main objectives in this study. Firstly, to determine whether the recombinant rat 6-phosphofructo 2-kinase (6PF2K, EC 2.7.1.105) and fructose 2,6-bisphosphatase (FBPase2, EC 3.1.3.11) enzymes, which catalyse the synthesis and degradation of Fru 2,6-P2 respectively, showed any catalytic activity as fusion proteins. Secondly, to alter the levels of Fru 2,6-P2 in sugarcane, an important agricultural crop due to its ability to store large quantities of sucrose, by expressing the recombinant genes. Thirdly, to investigate whether sugar metabolism in photosynthetic- (leaves) and non-photosynthetic tissue (internodes) were subsequently influenced. Activity tests performed on the bacterially expressed glutathione-S-transferase (GST) fusion 6PF2K and FBPase2 enzymes showed that they were catalytically active. In addition antibodies were raised against the bacterially expressed proteins. Methods for extracting and measuring Fru 2,6-P2 from sugarcane tissues had to be optimised because it is known that the extraction efficiencies of Fru 2,6-P2 could vary significantly between different plant species and also within tissues from the same species. A chloroform/methanol extraction method was established that provided Fru 2,6-P2 recoveries of 93% and 85% from sugarcane leaves and internodes respectively. Diurnal changes in the levels of Fru 2,6-P2, sucrose and starch were measured and the results suggested a role for Fru 2,6-P2 in photosynthetic sucrose metabolism and in the partitioning of carbon between sucrose and starch in sugarcane leaves. Transgenic sugarcane plants expressing either a recombinant rat FBPase2 (ODe lines) or 6PF2K (OCe lines) were generated. The ODe lines contained decreased leaf Fru 2,6-P2 levels but increased internodal Fru 2,6-P2 levels compared to the control plants. Higher leaf sucrose and reducing sugars (glucose and fructose) were measured in the transgenic plants than the control plants. The transgenic lines contained decreased internodal sucrose and increased reducing sugars compared to the control plants. Opposite trends were observed for Fru 2,6-P2 and sucrose when leaves, internodes 3+4 or internodes 7+8 of the different plant lines were compared. In contrast, no consistent trends between Fru 2,6-P2 and sucrose were evident in the OCe transgenic lines.