VTechWorks Repository :: Browsing by Author "Grabau, Elizabeth A."

Browsing by Author "Grabau, Elizabeth A."

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Antioxidant responses of pea (Pisum sativum L.) protoplasts
Doulis, Andreas G. (Virginia Tech, 1994-01-15)
Freshly isolated protoplasts from pea leaves were used to investigate the responses of antioxidant enzymes to oxidative stress. Two cultivars, Progress (tolerant) and Nugget (sensitive), that have differing resistance with respect to oxidative stress at the whole plant level were used. Sulfite and the superoxide generating herbicide, paraquat, were used as the oxidants. Final sulfite concentrations during photosynthetic incubations ranged from 1.5 mM to 30.0 mM. During the polarographic estimation of photosynthesis, CO₂-dependent O₂ evolution did not decrease. At sulfite concentrations of 3.0 mM or less, light-dependent O₂ evolution increased and was probably due to a concomitant SO₂-dependent O₂ evolution. Photosynthesis determined as ¹⁴CO₂ fixation was not increased at these low concentrations of sulfite. Concentrations greater than 7 mM = sulfite inhibited photosynthetic ¹⁴CO₂ fixation. No difference in these responses was found between the two cultivars. At 0.1 µM paraquat, the relative resistance to oxidative stress was reversed compared to previous studies at the whole plant level. With the tolerant cultivar, activity of the plastid antioxidant enzyme, glutathione reductase, increased after a three-hour exposure. Changes in the steady state level of glutathione reductase protein, as judged by immunoblots, did not correlate with the observed changes in enzyme activity. No change in the de novo synthesis of glutathione reductase occurred over the same period as a consequence of paraquat application. A mechanism, unrelated to oxygen free radical scavenging, may contribute to the relative tolerance to low concentrations of paraquat. On the other hand, after an eight-hour exposure to 0.1 mM PQ in the presence of Gamborg’s basal salts, superoxide dismutase activity of Progress protoplasts was enhanced 288% above the preexposure levels while glutathione reductase activity decreased 70% and ascorbate peroxidase activity decreased 90%. The relationship of these changes to oxidative damage to the photosynthetic machinery remains to be assessed.
Biosynthesis of Iron-Sulfur Clusters
Yuvaniyama, Pramvadee (Virginia Tech, 1999-11-17)
It is not known whether biosynthesis of [Fe-S] clusters occurs through a spontaneous self-assembly process or an enzymatic process. However, in the Azotobacter vinelandii nitrogenase system, it has been proposed that NifS and NifU are involved in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfur for the in vitro catalytic formation of [Fe-S] clusters. The activity of the NifU protein has not yet been established, but NifU could have functions complementary to NifS by mobilizing iron or serving as an intermediate site necessary for nitrogenase-specific [Fe-S] cluster assembly. A second iron-binding site within NifU was predicted to serve these functions because two identical [2Fe-2S] clusters that had previously been identified within the homodimeric NifU are tightly bound, and the NifU primary sequence is rich in cysteine residues. In this dissertation, I examined the possibility that NifU might mobilize iron or serve as an intermediate site for [Fe-S] cluster assembly, as well as the possibility that NifU could work in concert with NifS. Primary sequence comparisons, amino acid substitution experiments, and biophysical characterization of recombinantly-produced NifU fragments were used to show that NifU has a modular structure. One module is contained in approximately the C-terminal half of NifU and provides the binding site for the [2Fe-2S] cluster previously identified (the permanent [2Fe-2S] cluster). Cysteine residues Cys¹³⁷, Cys¹³⁹, Cys¹⁷⁵, and Cys¹⁷⁵ serve as ligands to the [2Fe-2S] cluster. Another module (referred to as NifU-1) is contained in approximately the N-terminal third of NifU and provides a second iron-binding site (rubredoxin-like Fe(III)-binding site). Cysteine residues Cys³⁵, Cys⁶², Cys¹⁰⁶>, and a putative non-cysteine ligand of unknown origin provide coordination to the iron at this site. The significance of these iron-binding sites was also accessed by showing that cysteine residues involved in providing the rubredoxin-like Fe(III)-binding site and those that provide the [2Fe-2S] cluster binding site are all required for the full physiological function of NifU. The two other cysteine residues contained within NifU, Cys²⁷² and Cys²⁷⁵, are neither necessary for binding iron at either site nor are they required for the full physiological function of NifU. These results provide the basis for a model where iron bound at the rubredoxin-like sites within NifU-1 (one iron per monomer) is proposed to be destined for [Fe-S] cluster formation. It was possible to find in vitro evidence supporting this idea. First, it was demonstrated that NifU and NifS are able to form a transient complex. Second, in the presence of NifS as well as L-cysteine and a reducing agent, the Fe(III) contained at the rubredoxin-like sites within the NifU-1 or NifU homodimer can rearrange to form a transient [2Fe-2S] cluster between the two subunits. Finally, a mutant form of NifU-1 was isolated that appears to be trapped in the [2Fe-2S] cluster-containing form, and this [2Fe-2S] cluster (the transient [2Fe-2S] cluster) can be released from the polypeptide matrix upon reduction with dithionite. Previous work has shown that the permanent [2Fe-2S] clusters of as-isolated NifU are in the oxidized form but can be reduced chemically. The transient [2Fe-2S] cluster formed between rubredoxin-like sites, in contrast, is reductively labile. If the transient cluster serves as an intermediate [Fe-S] cluster to be destined for [Fe-S] cluster assembly, I propose that the permanent [2Fe-2S] clusters could have redox roles participating in either one or all of the following events. The permanent [2Fe-2S] clusters could have a redox function in the acquisition of iron for initial binding at the mononuclear sites. They could also provide reducing equivalents for releasing the transient [2Fe-2S] cluster. In addition, upon releasing the transient [2Fe-2S] cluster, the permanent [2Fe-2S] clusters could provide the appropriate oxidation state of the irons to be destined to nitrogenase metallocluster core formation. Finally, because proteins homologous to NifU and NifS are widely distributed in nature, it is suggested that the mechanism for NifU and NifS in the formation of nitrogenase-specific [Fe-S] clusters could represent a general mechanism for [Fe-S] cluster synthesis in other systems.
Bulk segregant analysis for anther culture response and leptine content in backcross families of diploid potato
Boluarte, Tatiana (Virginia Tech, 1999-09-15)
Diploid potato populations between a primitive cultivated species, Solanum phureja, and a weedy species, S. chacoense, were used to examine the segregation of microsatellite markers and three traits in backcrosses. Two of the traits, anther culture competence and 2n pollen production, originated from S. phureja whereas the third, leptine production (a specific glycoalkaloid known to convey resistance to the Colorado potato beetle) originated from S. chacoense. Using CP2, a self-incompatible F₁ hybrid originating from a cross between S. chacoense clone 80-1 and S. phureja clone 1-3, three populations were developed: 1-3 x CP2 (PBCp), CP2 x 1-3 (PBCc), and CP2 x 80-1 (CBC). For the microsatellite study, four simple sequence repeat (SSR) primer pairs that amplified fragments within potato sequences found in the GenBank were used to look at segregation ratios in our backcross populations and to eliminate possible spurious genotypes bearing non-parental alleles in these populations. Seventeen spurious genotypes were discarded from PBCp; none was found in PBCc or CBC. Two SSR loci showed skewed segregation in PBCp (favoring transmissnion of the allele originally found in 80-1), PBCc showed normal segregation at all loci, and CBC showed distorted segregation at one locus (revealing a deficiency of homozygotes). In the study of anther culture, three components of ACR were investigated in a preliminary study: 1) embryos produced per anther (EPA), 2) embryo regeneration rate and 3) percentage of monoploids (2n=1x=12) among regenerants. CP2 was intermediate, 80-1 was low, and 1-3 was high for ACR. Only EPA was selected for further characterization in our populations. PBCp (78 genotypes) and CBC (57 genotypes), were characterized for anther culture response ACR/EPA in a series of studies. Nine high and ten low selections were identified in CBC, and ten high and ten low selections were identified in PBCp. EPA selections were used for bulk segregant analysis (BSA) using 214 RAPD primers. Two bands, one amplified by OPQ-10 and another by OPZ-4 were linked in coupling and in repulsion, respectively, to ACR in PBCp. One band amplified by OPW-14 primer was linked in coupling to ACR in CBC. One-way ANOVAs for data from remaining genotypes of the populations verified linkage of the markers to ACR/EPA. For 2n pollen production, a total of 77 PBCp genotypes was characterized; 80-1 produces low % 2n pollen, and 1-3 produces high % 2n pollen. Pollen samples were stained with propidium iodide and examined by flow cytometry. The frequency of 2n pollen varied continuously from 1.7 % to 40.6 % among the 41 genotypes that flowered sufficiently to allow three separate pollen collections. Variation due to the environment was observed where the frequency of 2n pollen appeared greater over a range of genotypes on single collection days. BSA could not be used due to limited population size and a low number of selections at the extremes of the distribution of phenotypes. The continuous variation for 2n pollen production suggests multigenic control of the trait. In the study of leptine content in reciprocal backcross populations, 87 genotypes within PBCp, and 42 genotypes within PBCc were characterized using gas chromatography of leaf samples. CP2 was intermediate, 1-3 had zero, and 80-1 was high for leptine content in the foliage. Leptines were present in low levels in 43 of 87 genotypes in PBCp, indicating simple genetic control. In PBCc, only 7 of 42 genotypes expressed leptines, generally at a higher level than in PBCp, indicating cytoplasmic inheritance. Ten high and ten nil selections within PBCp, and seven high and eight nil selections within PBCc were used for BSA using 214 RAPD primers. Three primers OPQ-2, OPT-16 and OPT-20 amplified bands segregating with high bulks in both populations. These markers were linked in coupling to leptine content in PBCp. Linkage was verified by ANOVAs for leptine content in the entire population.
cDNA Cloning and Gene Characterization of Large and Small Subunits of Ribonucleotide Reductase in Soybean
Xiong, Xinsheng (Virginia Tech, 1999-09-16)
Ribonucleotide reductase (RNR) reduces four ribonucleoside diphosphates to corresponding deoxyribonucleoside diphosphates, which are transformed into deoxyribonucleoside triphosphates, substrates for DNA polymerase. By controlling the supply and balance of deoxyribonucleoside diphosphates, RNR regulates DNA synthesis. RNR in E. coli and in animals consists of two identical large and two identical small subunits. Until recently, little was known about RNR in plants. For cloning RNR cDNA in plants, soybean (Glycine max) cDNAs were amplified with highly degenerate primers and the Rapid Amplification of cDNA Ends techniques. The cDNAs encoding two complete large subunits, one partial large subunit and one complete small subunit of RNR in soybean were cloned and sequenced. The RNR large subunits in soybean contain a motif with 20 amino acids, which appears to be specific for the RNR large subunits in plants. Southern hybridization results imply that a gene family encodes at least three different large subunits of RNR in soybean, and that a single gene encodes the small subunit. The presence of three different large subunits of RNR in soybean suggests that RNR complex in some plants may have a non-homodimer structure; alternatively, some plants may have different RNR isozymes. Northern hybridization results show that RNR large and small subunit genes in soybean are expressed both in dark-grown and light-grown seedlings, and that light does not increase RNR mRNA levels. Multiple poly(A) sites and different lengths of the 3â untranslated regions were found in cDNAs encoding some subunits of RNR in soybean. The same cis-acting elements may imprecisely locate some multiple poly(A) sites in plants.
Characterization of AgaR and YihW, Members of the DeoR Family of Transcriptional Regulators, and GlpE, a Rhodanese Belonging to the GlpR Regulon, Also a Member of the DeoR Family
Ray, William Keith (Virginia Tech, 1999-08-02)
AgaR, a protein in Escherichia coli thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for agaR, agaZ and agaS, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which agaR had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. In vivo and in vitro analysis of the agaZ promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the aga promoters was less sensitive to catabolite repression in revertants capable of N-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the E. coli genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of sn-glycerol 3-phosphate metabolism in E. coli, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress P_glpK, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress P_glpK. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of sn-glycerol 3-phosphate in E. coli by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. K_m (SSO₃²⁻) and K_m (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent K_m of 34 mM when thiosulfate was near its K_m, suggesting thioredoxin may be a physiological substrate.
Characterization of cytoplasmic diversity in soybean (Glycine max L. Merr) using mitochondrial markers
Hanlon, Regina (Virginia Tech, 1994-12-05)
Soybean, Glycine max L. Merr, is used worldwide as an important source of protein and oil for a wide spectrum of edible feed and industrial purposes. Modem cultivars are derived from relatively few plant introductions (PIs) which severely limits diversity in soybean germplasm. The United States Department of Agriculture (USDA) maintains the soybean germplasm collection. Mitochondrial DNA sequences have been used as markers of diversity at the cytoplasmic level. This project included three objectives. The first was a classification of the 208 varieties of the USDA's 'old domestic collection' of soybean varieties with two mitochondrial restriction fragment length polymorphisms (RFLP) markers. Molecular techniques were used to search for additional sources of cytoplasmic diversity available to soybean breeders. The two polymorphic markers were, a 2.3 kb HindIII fragment isolated from 'Williams 82' mitochondrial DNA, and a portion of the mitochondrial atp6 gene. These markers were used to distinguish cytoplasmic groups based on hybridization analysis of HindIII-digested soybean DNA Four major groups were observed with the 2.3 kb marker in the old domestic collection, and several minor subgroups were also detected. The second objective included subcloning and sequencing the 0.9 kb and 1.7 kb HindIII-PstI clones flanking the 2.3 kb HindIII fragment from 'Williams 82' DNA The total 4.9 kb PstI sequence from 'Williams 82' mitochondrial DNA was used to search a sequence database for any homology to known mitochondrial sequences. The third objective compared restriction maps of the four cytoplasmic types in the regions containing homology to the 4.9 kb PstI fragment. DNAs from the four cytoplasmic types were digested with five enzymes and four specific clones (0.9 kb PstI-HindIII, 0.8 kb HindIII-XbaI, 1.5 kb Xbal-HindIII, 1.7 kb HindIII-PstI) were used as hybridization probes in Southern analysis to examine RFLP patterns and construct comparative restriction maps of the four cytoplasmic types of DNA.
Characterization of Soybean Germplasm with Modified Phosphorus and Sugar Composition
Maupin, Laura Marie (Virginia Tech, 2010-09-20)
The development of soybean [Glycine max (L.) Merr.] cultivars with modified phosphorus (P) composition has nutritional and environmental benefits, but poor seed germination and emergence presents challenges for commercial production. Different genetic mutations in two sources of germplasm, CX1834 and V99-5089, decrease the phytate and increase the inorganic phosphorus (Pi) content of seed. In V99-5089, a mutation in the D-myo-inositol 3-phosphate synthase 1 gene (MIPS1) also results in elevated sucrose content with a concomitant decrease in raffinose and stachyose content, further improving the nutritional value of soybean meal. Prior to the release of V99-5089-derived germplasm, germplasm with the MIPS1 mutation was characterized and compared to CX1834-derived germplasm to determine the effects of this mutation on agronomic and seed composition traits in multiple environments. The correlations between P and sugar seed composition traits were favorable for improving the nutritional composition of soybean. Lack of genotype Ã environment interaction for sugar traits allows for selection in one growing environment. Despite the significant genotype Ã environment interaction for phytate and Pi, lines with the MIPS1 gene could readily be distinguished from normal phytate lines, even in unfavorable environments. Phenotypic selection for seed Pi content was more effective than marker assisted selection with the Satt453 marker. The CX1834-derived lines were lower for phytate and higher for Pi content compared to the V99-5089-derived lines. The use of subtropical winter nursery environments for population development resulted in significant reductions in emergence of low phytate genotypes, skewing segregation ratios and prohibiting the analysis of agronomic traits. Emergence was significantly affected by genotype, environment, and the genotype Ã environment interaction in three emergence tests of advanced low phytate lines. Emergence of modified lines was reduced but some were in a range that would not prohibit commercialization of P modified cultivars. Yields of the best emerging lines were not significantly different from the control cultivars. The results of this study indicate that the development of commercial cultivars with the V99-5089-derived MIPS1 mutation is possible but breeders and producers must focus attention on emergence during population development and seed production to emphasize selection of lines with high emergence potential.
Characterization of Transgenic Peanuts Expressing Oxalate Oxidase for Governmental Approval of Their Release for Control of Sclerotinia Blight
Chriscoe, Shanna Marie (Virginia Tech, 2008-07-08)
Sclerotinia minor Jagger is a fungal pathogen of cultivated peanut (Arachis hypogaea L.) that can cause crop losses in excess of 50%. Fungicides are not completely effective at controlling the disease and can cost up to $311 per hectare for three applications. The ability to produce oxalic acid is necessary for the pathogenicity of some Sclerotinia spp. With little to no naturally occurring resistance to Sclerotinia blight in Arachis spp., a biotechnological approach was used to confer resistance to the disease. Peanut plants were transformed with a gene from barley encoding oxalate oxidase, an enzyme that degrades oxalic acid. Transformed peanuts showed resistance to S. minor and increased yields under disease pressure compared to the parental lines. Before the resistant varieties can be marketed, they must be reviewed and approved by the governmental regulatory system. Responsibility for regulation of transgenic plants in the U.S. is shared among the U.S. Department of Agriculture (USDA) through the Animal and Plant Health Inspection Service (APHIS), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). These agencies require several different data sets including molecular characterization and field studies before each transformation event can be commercialized. This project was designed to characterize three different transformation events, N70, P39 and W171. Molecular characterization included determination of insertion number, copy number, intactness of the expression cassette and stable inheritance of the transgene. N70 was found to have two insertions and two copies while W171 had one insertion with one copy. The P39 event has two insertions and two or more copies. Each of the three events was stable over multiple generations. Phenotypic comparisons of each transgenic line to the parent cultivar were carried out in field studies. Characteristics such as oxalate oxidase expression, yield and quality, hay quality, disease occurrence, aflatoxin content and plant height were assessed. Transgenic peanuts showed few differences from the parent cultivar other than resistance to Sclerotinia blight and yield under disease pressure. Outcrossing studies were completed to determine the rate and distance of cross pollination. Outcrossing rates in N70, P39 and W171 were less than 2.5% and occurred up to 19 rows or 17.4 m from the nearest transgenic row. The molecular characterization and field performance of N70, P39 and W171 have been assembled into a document to petition APHIS for determination of non-regulated status.
Development of New and Alternative Resources for Breeding Low Phytate Soybeans
Burleson, Sarah Ann (Virginia Tech, 2011-04-18)
Soybean (Glycine max (L.) Merr) cultivars with genetic reduction in seed phytate concentration will be a valuable resource to livestock producers both nutritionally and economically, as well as in the preservation of the environment. However, due to association with reduced seed germination and field emergence potential, low phytate (LP) soybean cultivars are not being advanced to commercialization. Soybean breeders of LP lines must adjust their techniques and resources in order to efficiently select LP germplasm. Reduced seed germination and field emergence potential associated with LP germplasm eliminates many individuals with the desired LP trait during early generation population development, making improvement of agronomic performance among LP lines difficult. Thus, development of resources for improved phenotyping, genotyping, and early generation population development will facilitate the commercialization of LP cultivars. Discovery of variation for field emergence potential among F2-derived LP families suggests the potential for using a pedigree method for early generation population development in LP germplasm, as it preserves diversity and allows selection for improved field emergence. Integration of markers into a pedigree method will be useful to breeder. An example is BARCSOYSSR_11_1495 with 90-93% selection efficiency in various V99-5089-derived populations. Another resource for improved LP selection is a time-efficient, high-throughput modified iron (Fe) colorimetric phytate assay that does not suffer from inorganic phosphorus (P) concentration interference. Together these tools will provide breeders more accurate selection of LP lines.
Diallel analysis of diplopodia ear rot resistance in maize and an assessment of the genetic variability of Stenocarpella maydis through isozyme analysis
Dorrance, Anne E. (Virginia Tech, 1995-12-05)
Diplodia ear rot (DER) of maize (Zea mays L.) caused by the fungus, Stenocarpella maydis (Berk.) Sutton has increased in incidence in localized fields over the past decade. My research focused on screening for resistance by examining the development of DER following inoculations prior to flowering, analyzing a diallel cross for DER resistance, and examining the genetic variability of the fungus from isolates collected from the U.S. and the Republic of South Africa. DER developed in maize following inoculations with a spore suspension prior to flowering in both greenhouse and field evaluations. A spore suspension gave a better differentiation of resistance responses than dried preparations of colonized millet, colonized ground popcorn, or kernels from a diseased maize ear, all applied in the whorl 10 to 15 days prior to flowering (V12 for inbreds), and natural occurrence of disease. General combining ability was significant for both 1994 and 1995 growing seasons in an analysis of the F₁ of the diallel cross, indicating that additive gene action may be responsible for resistance and could be introduced into commercial cultivars. Specific combining ability was significant in 1995 and indicates that dominant gene action or epistasis may play role in DER resistance. There were minimal numbers of isozyme polymorphisms found in my S. maydis collection. Two isolates were polymorphic for esterase, two isolates were polymorphic for hexokinase and malate dehydrogenase and one isolate was polymorphic for hexose kinase. Fungi that have limited isozyme polymorphisms often are biotrophs or fungi with formae speciales which are usually limited to one host. These groups of fungi usually have races and this may indicate that a gene-for-gene interaction exists. These findings suggest that i) the whorl inoculation separates genotypes into resistant, intermediate, and susceptible groupings; ii) additive gene action is predominant form of inheritance, and iii) there are few isozyme polymorphisms in the population of S. maydis sampled.
Exploring protein interactions and intracellular localization in regulating flavonoid metabolism
Bowerman, Peter A. (Virginia Tech, 2010-08-02)
The organization of biological processes via protein-protein interactions and the subcellular localization of enzymes is believed to be fundamental to many aspects of metabolism. Although this organization has been demonstrated in several systems, the mechanisms by which it is established and regulated are still not well understood. The flavonoid biosynthetic pathway offers a unique system in which to study several important aspects of metabolism. Here we describe a novel toolset of mutant alleles within the flavonoid biosynthetic pathway. In addition, we discuss the use of several of these alleles together with a number of emerging technologies to probe the role of subcellular localization of chalcone synthase, the first committed flavonoid biosynthetic enzyme, on metabolic flux, and to characterize a novel chalcone synthase-interacting protein. The over-expression of this interacting protein induces novel phenotypes that are likely associated with the production or distribution of auxin. Further, interaction analyses between recombinant flavonoid biosynthetic enzymes point to the possibility that post-translational modifications play an important role in promoting interactions.
Expression of Human Protein C in Transgenic Tobacco
Ni, Hao II (Virginia Tech, 1997-12-01)
Human Protein C (hPC) is a vitamin K-dependent serine protease that has a critical role in the naturally-occurring anticoagulant pathway. Upon activation of the zymogen by thrombin at the endothelial cell surface, the active form of hPC has anticoagulant activity in hemostasis due to its ability to inactivate factors Va and VIIIa. For biological activity, hPC requires several post-translational modifications including proteolytic cleavage, disulfide bond formation, b-hydroxylation, g-carboxylation, and N-linked glycosylation. Plants have the eukaryotic protein modifying mechanisms required for many human proteins and may provide a safe, cost-effective system for producing hPC on a large-scale basis. Tobacco (Nicotiana tabacum L.) is particularly well suited for use as a bioreactor for high-value recombinant proteins. Tobacco is one of the easiest plants to transform, it is an excellent biomass producer and can produce up to a million seeds from a single genetically engineered plant. Previous attempts to produce hPC in tobacco were limited by expression levels. The overall goal of the research was to develop transgenic plants that express hPC at higher levels. A cDNA encoding hPC was fused to an enhanced constitutive 35S promoter (35SDE ) and introduced into a plant transformation vector. The hPC construct was introduced into tobacco leaf disks using Agrobacterium tumefaciens-mediated transformation, and 30 transgenic plants were generated. Stable integration of the hPC gene construct into the tobacco genome and transgene copy number were determined by genomic Southern hybridization and segregation analyses. The majority of transgenic plants expressed the hPC transgene based on RNA analyses by northern hybridization. Plants utilizing the enhanced 35S promoter had equivalent levels of expression to previously generated hPC-containing plants. A variety of polyclonal and monoclonal antibodies raised against hPC were tested for detection of hPC standards and tobacco-synthesized hPC by western immunoblotting. Novel proteins in the size range of hPC heavy chain cross-reacted with anti-heavy chain hPC antibodies in 35SDE:hPC plants. Thus, plants may be capable of synthesizing hPC and proteolytically processing it to light and heavy chains. Although further experiments will be required to confirm the identity of these putative hPC proteins in tobacco, these result suggest that analyses of hPC expressed in plants have been limited by effective tools for detecting the hPC gene product rather than expression levels determined by the transgene promoter.
Farnesyltransferase: Gene Expression in Plants and Role in Plant Development
Zhou, Dafeng (Virginia Tech, 1997-03-14)
Protein farnesyltransferase (FTase, E. C. 2.5.1.21) post-translationally modifies regulatory proteins involved in controlling cell growth, division, and differentiation. Recently, a cDNA clone (PsFTb) encoding a pea (Pisum sativum) FTase b subunit was isolated. Initial studies led to the hypothesis that FTase plays a role in the regulation of plant cell division. To gain insight into FTase function in plants, a detailed study of the expression pattern of FTase genes was carried out. A cDNA (NgFTb) encoding the b subunit of tobacco FTase was cloned from a Nicotiana glutinosa cDNA library to initiate studies in tobacco. In tobacco BY-2 suspension culture, levels of NgFTb mRNA and FTase activity transiently increased at the early log phase of cell growth and rapidly declined before cells entered stationary phase. These data, along with inhibitor studies in the BY-2 system, support our hypothesis. To understand the expression and regulation of pea FTase subunit genes, 5'-upstream sequences of both pea FTase subunit genes (PsFTb and PsFTa) were cloned from a pea genomic library. The 5'-upstream sequence (~2 kb) of PsFTa was fused to GUS (b-glucuronidase) and GFP (green fluorescent protein) reporter genes and introduced into tobacco plants. This 2 kb upstream region appears insufficient to provide PsFTa promoter function. On the other hand, 3.2 kb of PsFTb 5'-upstream sequence expressed as a PsFTb:GUS construct is fully functional in transgenic tobacco plants. GUS expression was most prominent in actively growing cells supporting FTase involvement in plant cell cycle control. GUS activity was also found in mature and imbibed embryos but not premature embryos, consistent with the role of FTase in abscisic acid (ABA) signaling. An unexpected pattern of GUS activity, not correlated with dividing cells or ABA signaling, was also observed in the transgenic plants. GUS activity was detected in vascular bundles adjacent to actively-growing tissues and in regions that connect two organs, e.g., junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls. Auxin promotes PsFTb expression while light and sucrose inhibit expression. These spatial and temporal expression patterns strongly suggest that FTase has a broader role associated with regulation of nutrient transportation or allocation in plants.
The Genetic Basis of Phytate, Oligosaccharide Content, and Emergence in Soybean
Glover, Natasha M. (Virginia Tech, 2011-06-27)
Soybean [Glycine max (L.) Merr] is one of the U.S.'s most economically important crops due to the protein and oil content of seeds. The major storage form of phosphorus in soybean seeds is found in the form of phytate, but because of its negative nutritional and environmental impacts, seed phytate and raffinosaccharide content have been a recent focus of breeders and molecular geneticists. The soybean line CX1834 is a low phytate mutant known to have two low phytate QTLs on linkage groups (LGs) L and N. The first objective of this research was to determine the genetic basis of the low phytate trait in CX1834. By using the whole genome sequence, we identified two candidate multidrug resistance-associated (MRP) ABC transporter genes. Sequencing the genes from CX1834 and comparing them to the reference genome sequence revealed a single nucleotide polymorphism (SNP) in the MRP gene located on LG N (causing a stop codon), and a SNP mutation in the MRP gene located on LG L (causing an amino acid change from arginine to lysine). One major concern with low phytate soybeans is the low seedling emergence. The second objective was to undertake a population-wide study of emergence in the recombinant inbred population CX1834 x V99-3337, over two years and two locations. We found a positive correlation between phytate level and emergence, and that variation among year, location, genotypic class, year x genotypic class, and year x location interactions were significantly affecting emergence. V99-5089, in addition to being low phytate, has high sucrose and low raffinosaccharide content. This phenotype of V99-5089 has been previously determined to be due to a SNP mutation in its myo-inositol phosphate synthase (MIPS) gene located on LG B1. The third objective was to use the recombinant inbred population derived from CX1834 x V99-5089 to observe the combinations of all three mutations to see how the different alleles impact phytate and raffinosaccharide content. The individuals with all three mutations, as well as those with the two MRP mutations together had lower phytate than the other genotypic classes. However, these lines (all three mutations) had unexpectedly high stachyose.
Genome-wide transcriptome analyses of developing seeds from low and normal phytic acid soybean lines
Redekar, Neelam R.; Biyashev, Ruslan M.; Jensen, Roderick V.; Helm, Richard F.; Grabau, Elizabeth A.; Saghai-Maroof, Mohammad A. (2015-12-18)
Background Low phytic acid (lpa) crops are potentially eco-friendly alternative to conventional normal phytic acid (PA) crops, improving mineral bioavailability in monogastric animals as well as decreasing phosphate pollution. The lpa crops developed to date carry mutations that are directly or indirectly associated with PA biosynthesis and accumulation during seed development. These lpa crops typically exhibit altered carbohydrate profiles, increased free phosphate, and lower seedling emergence, the latter of which reduces overall crop yield, hence limiting their large-scale cultivation. Improving lpa crop yield requires an understanding of the downstream effects of the lpa genotype on seed development. Towards that end, we present a comprehensive comparison of gene-expression profiles between lpa and normal PA soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. The lpa line used in this study carries single point mutations in a myo-inositol phosphate synthase gene along with two multidrug-resistance protein ABC transporter genes. Results RNA sequencing data of lpa and normal PA soybean lines from five seed-developmental stages (total of 30 libraries) were used for differential expression and functional enrichment analyses. A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. The results suggest that lpa-causing mutations play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively. Conclusions This study provides a global perspective of transcriptomal changes during soybean seed development in an lpa mutant. The mutants are characterized by earlier expression of genes associated with cell wall biosynthesis and a decrease in photosynthetic genes in late stages. The biological processes and transcription factors identified in this study are signatures of lpa-causing mutations.
Identification and Characterization of Genes Involved in Regulation of Ascorbate Metabolic Pathway(s) in Arabidopsis thaliana
Zhang, Wenyan (Virginia Tech, 2007-02-01)
Vitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination.
Identification and Characterization of Late Pathway Enzymes in Phytic Acid Biosynthesis in Glycine max
Stiles, Amanda Rose (Virginia Tech, 2007-07-30)
Phytic acid, also known as myo-inositol hexakisphosphate or Ins(1,2,3,4,5,6)P6, is the major storage form of phosphorus in plant seeds. Phytic acid is poorly digested by non-ruminant animals such as swine and poultry, and it chelates mineral cations including calcium, iron, zinc, and potassium, classifying it as an anti-nutrient. The excretion of unutilized phytic acid in manure translates to an excess amount of phosphorus runoff that can lead to eutrophication of lakes and ponds. Understanding the phytic acid biosynthetic pathway will allow for the development of low phytic acid (lpa) soybeans by the down-regulation of specific genes. The goal of this research was to elucidate the pathway(s) for phytic acid biosynthesis in soybean (Glycine max). We have isolated several myo-inositol phosphate kinase genes in soybean as possible candidates for steps in the biosynthetic pathway. We have characterized the genes for four myo-inositol(1,3,4)P3 5/6-kinases (GmItpk1-4), one myo-inositol(1,4,5)P3 6/3/5-kinase (GmIpk2), and one myo-inositol(1,3,4,5,6)P5 2-kinase (GmIpk1). We have examined expression in developing seeds and other tissues by Northern blot analysis and quantitative RT-PCR. We have expressed all six genes as tagged fusion proteins in E. coli, and verified enzyme activity on the proposed substrates. For each enzyme, we have conducted biochemical characterization to determine enzyme kinetics and substrate specificities. We have verified in vivo activity of GmIpk2 and GmIpk1 by complementing yeast mutants in the respective genes. Our studies indicate the likelihood that three of the genes may be involved in phytic acid biosynthesis: GmItpk3, GmIpk2 and GmIpk1. For future work, to more fully understand the contribution of each kinase gene to phytic acid biosynthesis, an RNA interference approach will be employed. The gene sequences identified in this study will be used to construct silencing vectors for use in future transformation of soybean embryogenic cultures to determine the effects of down-regulation on myo-inositol phosphate profiles.
Investigating R gene evolution by meiotic recombination using synthetic gene clusters in Arabidopsis
Sun, Jian (Virginia Tech, 2008-04-30)
Plant gene families organized as linked clusters are capable of evolving by a process of unequal crossing-over. This results in the formation of chimeric genes that may impart a novel function. However, the frequency and functional consequences of these unequal cross-over events are poorly characterized. Plant disease resistance genes (R genes) genes are frequently organized as gene clusters. In this study, I constructed an elaborately designed reconfigurable synthetic RPP1 (for resistance to Paranospora parasitica) gene cluster (synthRPP1) to model R gene evolution by meiotic recombination. This experimental design utilizes gain-of-luciferase phenotype (luc+) to identify and isolate recombinant R genes and uses two alternatively marked alleles to distinguish and measure different types of meiotic recombination (intra- vs. inter-chromosomal). Two putative single copy transgenic plants containing the synthRPP1 gene cluster were generated. These synthRPP1 gene clusters were reconfigured in vivo by two kinds of site-specific recombination systems (CRE/Lox, FLP/FRT) to generate two alternative versions of the synthRPP1 gene clusters in vivo. These lines, as well as others being developed, will be used in future genetic crosses to identify and characterize plants expressing chimeric RPP1 genes. My second area of research was to use a previously developed synthetic RBCSB gene cluster (synthRBCSB) gene cluster to investigate the relative frequency of meiotic unequal crossing over between paralogous genes located on either homologous chromosomes (homozygous lines) or sister chromatids (hemizygous lines). In contrast to published somatic recombination frequencies using a different reporter gene system, no statistically significant difference of meiotic unequal crossing over was observed between homo- and hemi-zygous synthRBCSB lines. This result suggests that meiotic unequal crossing-over between paralogs located on homologous chromosomes occurs at about the same frequency as paralogs located on sister chromatids. To investigate the rate of somatic recombination in synthRBCSB lines, a QRT-PCR method was developed to estimate the frequency of somatic recombination. Preliminary results suggest that the somatic recombination frequency was about 10,000 fold higher than meiotic recombination in the same generation. Moreover, two of five cloned chimeric genes that formed by somatic recombination indicated a different distribution of resolution sites than those observed in meiotic recombination. This finding suggests there are significant differences in both the frequency and character of somatic versus meiotic unequal crossing-over between paralogous genes in Arabidopsis.
Investigation of Transcriptional Regulation of 5'-Nucleotidase in Dictyostelium Discoideum
Eristi, Can M. (Virginia Tech, 2003-07-22)
A 5' AMP-degrading activity appears during the course of development in Dictyostelium discoideum between the prestalk and prespore zones. This enzyme is referred as 5'-Nucleotidase (5NT). Given the critical role of cyclic AMP in cell differentiation in this organism, 5NT is thought to be involved in cell positioning during development. Southern blot analysis showed a single form of the gene. The expression of the 5nt gene is known to be developmentally regulated. The message appears first at about 5 hr of the Dictyostelium development and remains constant throughout the rest of the development. Primer extension indicated two potential transcriptional start sites (118 bp and 148 bp upstream of the ATG initiation codon) for the 5nt expression. The 5nt promoter region was cloned and analyzed to investigate the expression of 5nt. Analysis of the cloned 5nt promoter fused to lacZ enabled the localization of the 5nt expression in pstAB cells during development. To identify cis-acting regulatory sequences, a series of 5' and internal promoter deletions were generated and fused to a luciferase reporter gene. The reporter activity driven by the 1,212 bp promoter started at the early aggregation stage, in agreement with temporal expression of the 5nt gene. Also, the expression was induced by exogenous cAMP. The reporter activity was high and relatively equivalent for all deletion constructs that contained 547 bp or more of the promoter region. No luciferase activity was detected using 365 bp or less of the promoter. A gradual decrease in activity was observed when three deletion constructs between -547 and -365 bp were tested suggesting the presence of at least two cis-regulatory elements within this region. Internal deletion analysis indicated another potential regulatory region located between -307 and -226 bp. To identify protein factor(s) that bind specifically to these regulatory sequences, gel shift assays were performed. Two bands, 0.33 Rf and 0.13 Rf, were detected in both cytoplasmic and nuclear extracts using radiolabeled DNA fragments located between -227 and -198 bp and -252 and -203 bp of the promoter region, respectively. Competition experiments confirmed the specificity of binding. The protein factors in these DNA binding activities were purified using various chromatography techniques. Mass spectrometry analysis of the purified 70 kDa protein corresponding to the 0.33 Rf band activity and a subsequent search in the Dictyostelium genomic database revealed that the purified protein was a putative formyltetrahydrofolate synthase.
Isolation and Characterization of D-Myo-Inositol-3-Phosphate Synthase Gene Family Members in Soybean
Good, Laura Lee (Virginia Tech, 2003-07-18)
The objective of this research was to isolate genes encoding isoforms of the enzyme D-myo-inositol 3-phosphate synthase (MIPS, E.C. 5.5.1.4) from soybean and to characterize their expression, especially with respect to their involvement in phytic acid biosynthesis. A MIPS-homologous cDNA, designated GmMIPS1, was isolated via PCR using total RNA from developing seeds. Southern blot analysis and examination of MIPS-homologous soybean EST sequences suggested that GmMIPS1 is part of a multigene family of at least four similar members. The sequences of promoter and genomic regions of GmMIPS1 and GmMIPS2 revealed a high degree of sequence conservation. Northern and western blot analyses showed that MIPS transcript and protein are abundantly expressed early in seed development. Immunolocalization of MIPS protein in developing seeds confirmed expression of MIPS early in seed development and correlated MIPS protein accumulation in soybean seed tissue with tissues in which phytic acid is known to accumulate. The promoter region of GmMIPS1 was isolated and analyzed for possible seed-specificity using promoter:GUS fusions. Two GmMIPS1 promoter fragments were capable of conferring GUS expression when bombarded directly into developing soybean seeds. However, preliminary bombardment experiments into soybean cell suspension culture indicated that both promoter fragments drove expression of GUS in undifferentiated tissue, indicating a potential lack of seed-specificity.

Browsing by Author "Grabau, Elizabeth A."

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