ssp-1 is a seed storage protein mutant which is deficient in one of the major seed storage proteins in Arabidopsis thaliana, the 12S cruciferins. To determine if this mutant can drive a higher level expression of a transgene than that found in wild type, the mutant was transformed with the phytohemagglutinin (PHA) gene and single copy PHA homozygotes were identified. These PHA transformants were crossed to wild type so that each PHA gene would be in the same copy number and chromosomal context in a wild type background. Immunoblotting was employed to compare the PHA levels of the single copy transformants in both genetic backgrounds. PHA levels ranged from 4.52% to 7.7% of the total protein in transformants. Two of the transformants showed 30.33% and 44.18% more PHA than that of their backcross. Therefore, a mutant such as ssp-1 may provide a means for overexpression of foreign proteins.

An interaction was recently detected between the potyviral protein, genome-linked (VPg) and the Arabidopsis thaliana translation initiation factor eIF(iso)4E (Wittmann et al., 1997). / Here, experiments were undertaken to address biological aspects of the VPg-eIF4E interaction. First, coimmunoprecipitation experiments performed with purified recombinant proteins have shown that VPg not only associates with eIF4E, as was previously published, but also with the larger eIF4F complex, of which eIF4E is a subunit. These results were confirmed by ELISA-type binding assays. It was also shown that there is no direct interaction between VPg and the other subunit of eIF4F, namely eIF4G. Finally, with the same experimental system, it was shown that the presence of eIF4G does not influence the binding affinity of VPg and eIF4E. / The interaction of VPg with the plant translation apparatus suggests that potyviral infection may alter the host protein expression profile. This hypothesis was investigated with the use of a protoplast system. We have shown that the global rates of protein synthesis in protoplasts transfected with an infectious TuMV cDNA clone dropped shortly after transfection, by as much as an estimated 70%. Recovery to normal levels occurred within 48 hours. / Evidence was obtained that the interaction between VPg and eIF4E is instrumental in this transient down-regulation of protein expression: protoplasts transfected with a mutant TuMV cDNA clone, the VPg of which has no affinity for eIF4E, failed to exhibit the drop in protein synthesis observed with the wild-type clone.

Turnip mosaic potyvirus (TuMV) is a single-stranded RNA plant virus. One of its proteins, the cylindrical inclusion (CI) protein, was hypothesized to interfere with host transcription via interaction with histone H3 protein. Interaction between CI and histone H3 was previously observed in Dr. Fortin's laboratory. Based on previous studies that demonstrated the importance of the H3 tail domain in gene regulation and chromosome arrangement, it was hypothesized that CI would interact with the tail rather than the globular domain. The objective of this project was to identify which histone H3 domains CI protein interacts with. The full-length, globular, and tail domains of histone H3 DNA were expressed in E. coli and purified. Based on in vitro interaction experiments, the CI protein was observed to interact with the globular domain of histone H3.

Cytoplasrnic male sterility (CMS) is a maternally inherited defect in the production of pollen, the male gamete of the flower. This sterility can be suppressed by nuclear Restorer of Fertility (Rf) genes that normally downregulate the expression of the CMS-associated novel mitochondrial gene. In Brassica napus, nap CMS and pol CMS are associated with related chimeric mitochondrial genes orf222 and orf224, respectively. CMS in both nap and pol is associated with a polar loss of locule development, loss of synchronous locule development and clumping of sporogenous tissue away from the tapetal cell layer, as well as secondary effects on petal and bud formation. In nap CMS, early accumulation of orf222 transcripts in the locule regions of developing anthers is associated with sterility, while the absence of orf222 transcripts from the locules is associated with fertility restoration. Accumulation of novel antisense transcripts of atp6 in a cell specific manner which matches that of sense transcripts of orf222 and atp6 in nap CMS anthers may be indicative of a post-transcriptional regulatory mechanism associated with CMS in flower buds. / Restoration of fertility in Brassica napus nap and pol CMS is associated with nuclearly encoded genes Rfn and Rfp, respectively. These restorers are very closely linked to one another, and may be allelic. Further efforts to isolate Rfp have narrowed the genomic region to approximately 105 kb of a syntenic region in Arabidopsis thaliana. Cosmid clones isolated from a library of Brassica rapa genomic DNA introgressed with Rfp have been successfully sorted into contigs through the application of the amplified fragment length polymorphism technique. The region to which Rfp is mapped is syntenic to a region of Arabidopsis DNA that is a duplication of a second location at the 23 megabase region of chromosome 1 of that genome. This region contains pentatricopeptide (PPR) motif-encoding genes that are highly related to other restorers of fertility of other species. By inference, Rfp from Brassica napus may encode PPR motifs. The PPR genes related to these previously characterized restorers of fertility are often found alongside the restorer genes existing as mini-clusters of several PPR-encoding genes. This is likely caused by selective pressure acting on PPR-encoding genes that resulted in diversification and multiplication of these genes. In addition, the PPR genes of this duplicated region are not syntenically located, whereas the non-PPR-encoding genes maintain their syntenic locations. The same is true for orthologous comparisons between Arabidopsis and other plant species. PPR genes are therefore malleable and capable of alteration in response to changing environmental pressures, such as the evolution of sterility inducing genes.

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.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

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

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 15, 2008) Vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 3 uppsatser.

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