Browsing by Author "Flinn, Barry S."

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    Biosynthesis of Steroidal Glycoakaloids in Solanum chacoense Bitter
    Mweetwa, Alice Mutiti (Virginia Tech, 2009-07-24)
    Steroidal glycoalkaloids (SGAs) are secondary metabolites produced by approximately 350 species in the Solanaceae family. SGAs are reported to be important for pest resistance and flavor enhancement at low concentrations but are toxic to humans and other mammals at high concentrations. Studies on sterol / SGA biosynthesis have implicated squalene synthase as a key regulatory enzyme because it catalyzes an irreversible step from the mevalonic acid pathway. However, the regulatory mechanisms of squalene synthase are not yet known. A study was conducted to elucidate the distribution pattern of SGAs and to clone the squalene synthase gene in order to determine a relationship between SGAs and gene expression levels. Solanum chacoense, a wild potato species was used as a model plant from which tissues were harvested at specified developmental stages and analyzed for SGA content. The results from the SGA analysis suggest a qualitative and quantitative tissue- and age-dependent accumulation of SGAs. Regenerative tissues such as, axiliary shoots, flowers and floral buds had the highest levels of 88, 49 and 63 µmole/g DW, respectively. The roots, stems and tubers showed the lowest amounts of SGAs of 1 to 8, 5 to 15 and 7 to 15 µmole/g DW, respectively. Stolons and tubers accumulated higher amounts of α-chaconine (59 to 67%) than α-solanine (61 to 64%) at all developmental stages analyzed. On the other hand, in young expanding, fully expanded, and old senescing leaves where leptine and leptinines tend to dominate, α-solanine and α-chaconine together accounted for only 8 to 15%, 7 to 15%, and 8 to 45%, respectively. Plant organs that showed the highest biosynthetic activity for SGA production also had high levels of transcripts coding for genes of isoprenoid biosynthesis. The results from the cloning and characterization of squalene synthase suggest that the cloned cDNA fragment is a putative S. chacoense squalene synthase gene with an open reading frame / predicted protein precursor of 411 amino acids. The cloned cDNA has high similarity (68-100%) to known plant squalene synthase genes and contains six deduced peptide domains observed in other species. The 3â untranslated regions of floral buds, young leaves (early vegetative stage), and fully expanded leaves (anthesis) were different in length with, 249, 335, and 202 nucleotides, respectively. The Southern blot analysis suggests a single copy gene although the existence of a gene family cannot be ruled out.
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    Characterization of activation tagged potato (Solanum tuberosum L.) mutants
    Aulakh, Sukhwinder Singh (Virginia Tech, 2012-09-14)
    Generation and characterization of activation tagged potato mutants could aid in functional genomic studies. Morphological and molecular studies were conducted to compare potato cv. Bintje, its two mutants, underperformer (up), and nikku generated using the activation tagging vector pSKI074, and nikku revertant plants. Mutant up exhibited a dwarf phenotype (plant height 42 cm vs. 73 cm in cv. Bintje), abundant axillary shoot growth (3.1 shoots/plant compared to 0.7 shoots/plant in cv. Bintje; in vitro plants), greater tuber yield, altered tuber traits and early senescence compared to wild-type Bintje under in vitro conditions. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant were consistent, but the tuber yield of up was less (250 g/plant compared to 610 g/plant in wild-type Bintje) and had fewer axillary shoots compared to wild-type (1.9 shoots/plant in up vs. 4.7 shoots/plant in Bintje). Mutant nikku plants exhibited an extremely dwarf phenotype (plant height 2 cm in nikku vs. 6 cm in Bintje), had small hyponastic leaves, were rootless, and infrequently produced small tubers when compared to cv. Bintje. The overall nikku phenotype was suggestive of a constitutive stress response, which was further supported by the higher expression levels of several stress-responsive genes in nikku. The nikku revertant plants exhibited near normal stem elongation, larger leaves and consistent rooting, and it was a case of partial reversion. Southern blot analyses indicated the presence of single T-DNA insertions on chromosome 10 in the up and on chromosome 12 in the nikku mutant. The reversion in the nikku plants was not associated with the loss of enhancer copies from the original nikku mutant. Reverse transcriptase PCR analyses indicated transcriptional activation/repression of several genes in the up and nikku mutants, suggesting pleiotropic effects. In revertant, the expression levels of several genes which were differentially regulated in the nikku mutant were similar to Bintje. The gene immediately flanking the right border of the T-DNA insertion, which encoded a novel BTB/POZ (Broad complex, Tramtrac, Bric a brac; also known as Pox virus and Zinc finger) domain-containing protein, was highly up-regulated in the up mutant. This protein domain plays an important role in several important developmental, transcriptional and regulatory pathways. The mRNA-seq analyses resulted in 1,632 genes that were differentially expressed between mutant up and Bintje and the total number of up-regulated genes (661) were less than the number of genes down-regulated (971 genes) in the up mutant. Further analyses indicated that a variety of biological processes including decreased cell division, cell cycle activity, and abiotic stress responses were modified in the up mutant. In the nikku mutant, two potato genes, encoding an Acyl-CoA N-acyltransferases (NAT) superfamily protein, and a predicted major facilitator superfamily protein (MFS) were identified and overexpression lines Bintje/35S::NAT1 and Bintje/35S::PMT1 were created for recapitulation of the nikku mutant phenotype. Methylated DNA-PCR between the nikku and the revertant indicated a change in methylation status of the 35S enhancers, suggesting that the nikku revertant phenotype may be associated with some epigenetic modification.
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    Identification and characterization of a matrix metalloproteinase (Pta1-MMP) expressed during Loblolly pine (Pinus taeda) seed development and germination
    Ratnaparkhe, Supriya M. (Virginia Tech, 2009-03-19)
    Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. The Pta1-MMP protein has the structural characteristics of other plant MMPs, and a recombinant protein (rPta-MMP) generated by using EST sequences for a seed-expressed MMP exhibits Zn2+-dependent protease activity, and is inhibited by the active site-binding hydroxamate inhibitor GM6001 and EDTA. The Pta1-MMP gene is expressed during embryo development, with transcript levels increasing from proembryo to early cotyledonary stage, then declining during late cotyledonary expansion and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed imbibition in water facilited germination, which was stimulated by GA3 and inhibited by ABA. The timing of germination was mirrored by the presence of MMP-like protease activity in both water- and GA3-imbibed embryos. Pta1-MMP transcript levels increased in association with germination for both GA3- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 10 days after imbibition, Pta1-MMP transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed imbibition inhibited germination in a dose-dependent manner. Our results suggest that Pta1-MMP is required for ECM modification, facilitating the cell division and expansion required for both embryo development and germination. To our knowledge, this is the first report of an MMP in any gymnosperm and also its involvement in embryo development and subsequent germination.
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    Somatic embryogenesis in southern and tropical pine species: Loblolly pine (Pinus taeda), Longleaf pine (P. palustris) and Oocarpa pine (P. oocarpa)
    Lara-Chavez, Alejandra M. (Virginia Tech, 2010-08-11)
    The focus of the current project was to establish an improved and reliable protocol for somatic embryogenesis in 1) Pinus taeda and Pinus palustris; pine species of high value for commercial applications and germplasm conservation supported through breeding programs at The Virginia Department of Forestry (Chapter III); and 2) Pinus oocarpa; an economically important pine species in the southern half of Mexico and Central America (Chapter IV). In addition, 3) the study of the gene expression analysis of developmental stages of both somatic and zygotic embryos of P. taeda was compared to assess developmental fidelity at the molecular level (Chapter V). By testing four basal media combined with different plant growth regulator combinations, we have established stable embryogenic cultures from high value families of P. taeda and P. palustris using the tissue culture medium 1218 (Pullman et al 2005) in combination with an auxin:citokinin ratio at 10:5 (molar). However, optimization of the protocols for the maturation and further conversion of somatic embryos to seedlings requires further work. For P. oocarpa, we hypothesized that somatic embryo induction may be possible by mimicking natural seed-embryo developmental conditions, and a new tissue culture medium, based on the mineral content of the seed nutritive tissue (megagametophyte), was formulated. The novel culture medium (PO) was tested in combination with different plant growth regulator concentrations for the initiation of somatic embryogenesis from fresh collections of P. oocarpa immature zygotic embryos. Additionally, the established embryogenic cultures were able to mature and germinate, to our knowledge resulting in the first report of the production of P. oocarpa plantlets through somatic embryogenesis. PO medium also has the potential to be used successfully for other tropical pine species which today suffer from suboptimal somatic embryogenesis protocols. The fundamental study of molecular regulation of embryo development showed that under the current maturation conditions, P. taeda somatic embryos were temporally similar in gene expression to zygotic embryos of the same species. However, potentially important differences were found and results could potentially explain the low germination success during somatic embryogenesis. More research is still needed to further explore the natural environment of developing seed embryos to improve the somatic embryogenesis protocols and to enable full integration of this clonal propagation method into the breeding programs for pines.