Browsing by Author "Arthur Weissinger, Committee Member"

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Coenzyme Q10 Biosynthesis in Plants: Is the Polyprenyltransferase an Appropriate Gene Target for the Increased Production of CoQ?
(2010-09-02) Stiff, Michael; David Danehower, Committee Chair; Arthur Weissinger, Committee Member; Jose Alonso, Committee Member; Nigel Deighton, Committee Member; John Williamson, Committee Member
The Effects of T-DNA Integration Sites on Transgene Expression in Arabidopsis
(2004-06-28) Francis, Kirk Eric; Mark Conkling, Committee Member; William F. Thompson, Committee Co-Chair; Steven Spiker, Committee Chair; Arthur Weissinger, Committee Member
Several recent investigations of T-DNA integration sites in Arabidopsis thaliana have reported "cold spots" of integration, especially near centromeric regions. These observations have contributed to the ongoing debate over whether T-DNA integration is random, or if integration preferentially occurs in transcriptionally active regions. When transgenic plants are identified by selecting or screening for transgene activity, transformants with integrations into genomic regions that suppress transcription, such as heterochromatin, may not be identified. This phenomenon, which we call selection bias, may explain the perceived non-random distribution of T-DNA integration in previous studies. In order to investigate this possibility, I have characterized the sites of T-DNA integration in the genomes of transgenic plants identified by pooled PCR, a procedure that does not required expression of the transgene and is therefore free of selection bias. Over 100 transgenic Arabidopsis plants were identified by PCR and compared to kanamycin-selected transformants from the same T1 seed pool. A higher perceived transformation efficiency and a higher frequency of transgene silencing were observed in the PCR-identified lines. Together, the data suggest approximately 30% of transformation events may result in non-expressing transgenes that would preclude identification by selection. Genomic integration sites in PCR-identified lines were compared to those in existing T-DNA integration databases. In PCR-identified lines with silenced transgenes, the integration sites mapped to regions significantly underrepresented by T-DNA integrations in studies where transformants were identified by selection. The data presented here suggest that selection bias can account for at least some of the observed non-random integration of T-DNA into the Arabidopsis genome.
Plant Responses to Stress in Acid Environments: An Assessment of the Role of Mycorrhizal Fungi.
(2006-08-16) Moyer-Henry, Kari Anne; Thomas Rufty, Committee Chair; Nina Allen, Committee Member; Barry Goldfarb, Committee Member; Arthur Weissinger, Committee Member
The purpose of the research was to gain further understanding of the responses of plants to stress in acid environments and the role of mycorrhizal fungi in stress tolerance. Acidic soils of the southeastern coastal plain typically have low fertility, but weeds remain problematic even in soybean and peanut fields where no fertilizer nitrogen is applied. Field experiments using ¹⁵N natural abundance examined whether nitrogen might be transferred between the N₂-fixing crop species and neighboring weeds. A five year field study demonstrated that substantial nitrogen transfer did occur. Because nitrogen transfer was largely dependent on the presence of arbuscular mycorrhizae, the results strongly suggested that N moved from plant to plant through mycorrhizal hyphae that connected plant root systems. Another characteristic of acid soils is the presence of high levels of aluminum. When soil pH is below 5.0, the presence of Al⁺³ in soil solution can cause aluminum toxicity to occur in many plants. Aluminum toxicity inhibits root growth and predisposes crop plants to drought and nutrient deficiencies. Loblolly pine was determined to be extremely tolerant to aluminum. In a series of experiments, we examined the ability of loblolly pine to exclude aluminum from root tip meristems which are known to be the main sites of aluminum toxicity. The primary and secondary roots of pine exhibited high degrees of Al tolerance. Tolerance was associated with Al exclusion from the root tips and, of the Al accumulating in the root, exclusion from the root meristem. Ectomycorrhizal colonization was found to contribute to aluminum tolerance, evidently by providing an extra barrier to Al entry into the root. Additional experiments examined Al relations of arbuscular mycorrhizal fungi. The root systems of more than 80% of all plant species are colonized by arbuscular mycorrhizae. In the presence of aluminum, colonization by arbuscular mycorrhizal fungal species was inhibited. The results suggested alterations in root function caused lower colonization, as mycorrhizal infection potential appeared unaffected. Aluminum also inhibited fungal spore germination, but only at very high Al levels. Much of the function of the mycorrhizal fungi was unaffected even as aluminum accumulated in fungal structures.
The Use of Flow Cytometry to Investigate the Effects of Matrix Attachment Regions on Transgene Expression in Plant Cells.
(2005-02-14) Halweg, Christopher Jay; Arthur Weissinger, Committee Member; William Thompson, Committee Co-Chair; Steven Spiker, Committee Co-Chair; Dominique Robertson, Committee Member
Many studies in both plant and animal systems have shown that Matrix Attachment Regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. MARs are AT-rich sequences of DNA that bind in vitro to the proteinacous filament-like structure within the nucleus called the nuclear matrix. In our investigation of transgenic Nicotiana tabacum NT-1 cells in culture, we have observed that transgene expression is often variegated. In other words, some cells in an isogenic population do not express the transgene, and/or other cells within the same population express the transgene at varying levels. The question was raised: Do MARs increase transgene expression by altering variegation? More specifically, do MARs increase the percentage of cells expressing the transgene, increase the magnitude of expression in expressing cells, or both? In order to address these questions, it was necessary to quantitate transgene expression variegation at the resolution of individual cells. We chose to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells by flow cytometry. In order to analyze individual cells and because NT-1 cells in culture grow as filaments, it is necessary to prepare protoplasts that can pass one at a time through the flow cell of the flow cytometer. We found that current flow cytometry methods for measuring GFP expression in plants were susceptible to debris resulting from protoplast preparations. We observed that when the plasma membrane of protoplasts is breached, GFP diffuses out into the medium, and flow cytometric measurements of these non-viable protoplasts imply they do not express GFP. This debris can overestimate the proportion of non-expressing cells in the population. In order to correct this problem, we used an approach called a dye exclusion test. Because propidium iodide enters protoplasts to stain nuclei only when the plasma membrane is breached, debris that stains with this dye can be removed from our analysis. Using this approach we were able to quantitate GFP expression in individual cells without complications from debris. We used flow cytometry to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells from lines transformed by Agrobacterium. We find that in this system the Rb7 MAR increases GFP expression 2-4 fold. This increase is caused by both an increase in the percentage of expressing cells and an increase in the magnitude of expression in expressing cells. Cell lines transformed with MAR-containing vectors averaged 27-39% more cells expressing GFP and these cells expressed GFP at 2-3 fold higher levels than cells transformed with control constructs. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from a population of cell lines obtained by liquid culture of entire Agrobacterium co-cultivation plates. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression. Our results indicate that the Rb7 MAR increases the frequency of transgene expression, presumably by reducing gene silencing, while also increasing the levels of expression in expressing cells, perhaps through an enhancer-like activity.