Two conditions of alkaline hydrolysis of proteins, (1) 2M NaOH, 18 hours, 100°C and (2) 3M NaOH, 16 hours 110°C, prior to ion-exchange chromatography were tested on free amino acids and model protein systems to determine the better set of conditions for measurement of methionine sulfoxide in food proteins. Recoveries of methionine, methionine sulfoxide, and methionine sulfone from base-hydrolyzed amino acid mixtures were, respectively, 89, 100, and 105% with the 2M NaOH conditions and 83, 90, and 98% with the 3M NaOH conditions. The percentages of total methionine, determined by performic acid oxidation, recovered as methionine, methionine sulfoxide, and methionine sulfone after hydrolysis with 2M NaOH were, respectively 101, 0, and 0% in lysozyme, 68, 25, and 0% in oxidized lysozyme, 74, 3, and 0% in casein and 0, 74, and 0% in oxidized casein. The presence of glucose in the hydrolysis mixture with the model proteins caused as much as 8% oxidation of methionine to methionine sulfoxide. The presence of copper (II) and iron (II) ions along with sugars did not increase the amount of methionine generated in casein and a soy isolate. Methionine sulfone was never generated in any of the model systems. These results suggested that determination of methionine sulfoxide after basic hydrolysis with 2M NaOH in foods low in carbohydrates is valid but in foods high in carbohydrates the procedure may slightly overestimate the methionine sulfoxide content. Acid hydrolysis of free methionine sulfoxide reduced 15% of the methionine sulfoxide to methionine while acid hydrolysis of oxidized lysozyme and oxidized casein led to reduction of all the methionine sulfoxide in these proteins. Eight food products were analyzed for methionine, methionine sulfoxide, and methionine sulfone. Total methionine was measured by the performic acid oxidation method, methionine sulfone by ion-exchange chromatography after acid hydrolysis, methionine sulfoxide by ion-exchange chromatography after hydrolysis with 2M NaOH for 18 hours at 100°C, and methionine by the difference between total methionine and the sum of methionine sulfoxide and sulfone. Only a trace of methionine sulfone and less than 6% of total methionine was present as methionine sulfoxide in a soy flour and a concentrate. Two soy isolates contained 74 and 8% of total methionine as sulfoxide and 6 and 4%, respectively, as sulfone. Two soy-based infant formulas contained 17 and 12% of total methionine as the sulfoxide and 12 and 8%, respectively, as sulfone. Two milk-based formulas contained 18 and 9% as sulfoxide and 8 and 13%, respectively, as sulfone. The feasibility of using ATP:L-methionine S-adenosyltransferase to determine the number of unaltered methionine residues in food proteins was also explored. Di- and tripeptides composed of methionine appeared to be able to function as well as L-methionine as substrates. Spectrophotometric studies suggested that the enzyme could act on methionine residues in two soy isolates; however, these results could not be confirmed by amino acid analyses of the isolates after incubation with ATP and the enzyme. / Ph. D.

This study investigated the nutritive value of Toasted Soy Protein (TSP) when it is incorporated in a basal rice diet. Forty-two male weanling rats were used for a period of 28 days. A total of five diets were fed to five groups of animals having seven animals in each group. An initial group of seven was also included. The composition of the five diets is: 6% rice protein (Diet l); 6% rice protein supplemented with 4% TSP (Diet 2); 6% rice protein supplemented with 4% TSP and 0.15% methionine (Diet 3); 6% rice protein supplemented with 4% casein (Diet 4); and 6% rice protein supplemented with 6.5% TSP. The growth rate for animals fed Diets 2, 3, 4, and 5 is similar, all animals showing an almost uniform weight gain. Supplementation of methionine to Diet 2 did not produce marked increase in weight gain. There is no significant difference in protein efficiency ratio (PER) among animals ingesting Diets 2, 3, 4, and 5. However, an increase of the TSP level from 4% to 6.5% reduced the PER slightly. There was a direct relationship between body weight gain and nitrogen retention as measured by nitrogen balance and liver nitrogen content. The methionine content of the diets and plasma was detennined by microbioassay. The total sulfur-containing amino acid content of Diet 2, i.e.: 0.478% is very close to the requirement of 0.5% for the growing rat. No direct relationship was found between methionine content of the diet and of the plasma. A combination of 6% rice protein supplemented with 4% TSP could be a protein of high quality and this mixture of proteins of plant origins can support and promote good growth of young rats. / Master of Science

A plethora of information has been amassed in attempts to understand the basic principles governing living systems. Since the birth of the modern scientific method, many compelling theories have been devised as scientists attempt to understand the mysteries of life. The endo-symbiont theory for the biogenesis of mitochondria and chloroplasts is one such theory that has recently gained increased in acceptance with the maturity of molecular and biochemical techniques. These two organelles are unique in that they contain DNA which codes for some of the proteins involved in organellar function. Maternal inheritance of some traits such as cytoplasmic male sterility (cms) in plants can be linked to mitochondrial DNA. However, further understanding of mitochondrial gene expression, regulation and DNA rearrangements has been hindered by the lack of a transformation system for these organelles. A longterm goal in the field is to develop a transformation and expression system for soybean mitochondria. Despite recent transformations of yeast mitochondria, no successful transformations of higher plant mitochondria have been reported to date. Mitochondrial transformation occurs at low frequencies, and therefore requires a method of selection, i.e. a means by which to differentiate cells containing a transformed mitochondrion from the background of largely untransformed cells. The work presented in this paper is the foundation for the future development of a selectable marker for plant mitochondrial transformation. The antibiotic oligomycin is an inhibitor of the mitochondrial ATP synthase complex, and therefore has potential for the isolation of a marker gene. Two approaches, soybean tissue culture mutagenesis and site-directed mutagenesis of the soybean [Glycine max (L.) Merr.] atp9 gene, have been explored in the hopes of isolating an oligomycin-resistant marker. In yeast (Saccharomyces cerevisiae), atp9 mutants have been shown to contain nucleotide mutations resulting in single amino acid substitutions that confer resistance to oligomycin. Sites for mutagenesis of the soybean atp9 gene were chosen based on DNA sequence homology between the yeast and soybean genes. Before site-directed mutagenesis, atp9 cDNA clones were cloned and sequenced, revealing RNA editing sites. Among these cDNA clones, we have also sequenced atp9 from an oligomycin-resistant soybean suspension culture for possible mutations in the gene. Suspension cultures were mutagenized with EMS and selected by growth inhibition with oligomycin. Inhibition curves showed an increased tolerance for oligomycin for EMS-treated cultures compared to wild-type. For future testing of the mutagenized atp9 gene we have constructed a particle inflow gun to introduce the gene(s) of interest into the nucleus of soybean suspension culture cells. A mitochondrial target sequence from β-atp has been fused in frame to the atp9 gene to direct the mutagenized gene product, now the product of a nuclear gene, correctly into the mitochodrion. / Master of Science

Soy protein concentrate (SPC) was autoclaved at 121°C and 15 p.s.i for 0, 10, 30 min., 2 and 4 hr. The solubility of the control, 10 and 30 min. autoclaved samples was quite low (about 12%) as compared with the 2 and 4 hr. samples (about 27%) in potassium phosphate buffer (pH 7.6, 0.5 ionic strength). When urea and beta-mercaptoethanol were added in increasing concentration to the buffer, the solubility of control, 10 and 30 min. samples increased dramatically up to 60%; while the solubility of 2 and 4 hr. autoclaved samples changed very little remaining at 25 to 40% throughout. Digestibility of SPC samples was determined by three in vitro methods, all including treatment of SPC with selected proteases, followed by measurement of (1) TCA soluble N production (2) breakdown products via SDS-PAGE and (3) pH drop resulting from enzyme action. In vivo apparent digestibility was determined in a rat feeding study. The digestibility of SPC samples were found to be significantly affected by length of autoclaving. The digestibility of control was higher than autoclaved samples as determined by both in vivo and in vivo assays. The digestibility of 10 and 30 min. autoclaved SPC samples was significantly higher than 2 and 4 hr. autoclaved samples in in vivo assays. While, apparent in vivo digestibility of 10, 30 min. and 2 hr. autoclaved samples was significantly higher than the 4 hr. sample. Correlation coefficients of in vivo digestibility as determined by the TCA soluble N measurement, SDS-PAGE and pH drop method with in vivo apparent digestibility were 0.96, 0.92 and 0.95, respectively. There was no actual destruction of amino acids except cysteine; 10, 30 min., 2 and 4 hr. SPC samples contained 6, 20, 27 and 39% less cysteine respectively than the SPC control. The chemical score of SPC samples indicated that cysteine became the first limiting amino acid in the 2 hr. sample. PERs of the 2 and 4 hr. autoclaved SPC samples were significantly less than control, 10 and 30 min. autoclaved samples. While C-PERs of control, 10 min. samples were higher than 30 min., 2 and 4 hr. samples. Decreased PER values of autoclaved SPC samples were likely due to i) decreased protein digestibility and rate of enzymatic hydrolysis, ii) destruction of essential amino acids, and iii) decreased food intake. / Ph. D.

Phytate is the major storage form of phosphorus in seeds of soybeans. Because phytate chelates mineral cations including calcium, iron, and zinc, these mixed salts are often excreted by non-ruminant animals such as humans, swine, poultry, and fish. While this causes iron and zinc deficiencies, phytate is also considered a water pollutant due to the excess phosphorus excreted in animal waste. These negative environmental and nutritional effects, create a need for low phytate soybeans. While several low phytate soybean lines have been developed, a major drawback is the reduced seedling emergence of these lines resulting in low yields. Therefore, understanding the genetic and molecular bases of the low emergence trait in relation to seed phytate content in major crops such as soybean is of great economic importance. This PhD project worked towards the long term goal of developing low phytate soybean cultivars with good seedling emergence and high-yield. This dissertation focused on metabolomic differences between low and normal phytate lines and how these could relate to the low emergence phenotype. The genetic materials used here include four near isogenic lines that differ in mutations in two multi drug resistance-associated proteins (MRPs). Only the line with both mutations was low phytate. The phytate levels, field- and lab-based emergence rates were determined for these lines, their parents and a control line through replicated field experiments for three consecutive years. The emergence rates of the low phytate lines were not always reduced. This showed that the environment the seeds were produced in is highly important, especially when breeding and commercially growing low phytate lines. A protocol was developed for successful metabolomic discrimination of these closely related soybean lines. The polar and non-polar metabolite profiles were determined using ultra performance liquid chromatography mass spectrometry and metabolomic differences between the low and normal phytate lines were identified. The low phytate double mutant did not contain C22 glucose terminated Group A soyasaponins and almost exclusively contained C22 xylose terminated Group A soyasaponins (A4, A5 and A6). Compared to the normal phytate lines, the low phytate soybean line showed a higher concentration of storage lipids (triacylglycerols and diacylglycerols) and certain phospholipids. / Ph. D.

Two Virginia isolates of soybean mosaic virus (SHV), VA and OCM, were classified as G1 and G3 strains, respectively, according to a soybean differential cultivar system. G1/VA and G3/OCM differed in the symptom severity induced on soybean (Glycine max [L.]Merr.) plants and by the production by G3/OCM of cytoplasmic strands, containing virus particles, within infected leaf cells. Two Illinois isolates, G1/IL and G3/IL, resembled G3/0CM in mild symptoms; induced in soybean plants and by the production of cytoplasmic strands ultrastructurally. G1/VA differed from the 3 mild strains by the severe symptoms induced on plants and by the absence of cytoplasmic strands ultrastructurally. All 4 strains were closely related serologically. The tolerant reaction of soybean to the 3 mild SMV strains was correlated with production of cytoplasmic strands. Soybean plants were most susceptible to G1/VA and peanut stunt virus (PSV) when the primary leaves were inoculated at 50-75% of their full expansion. Plants developed more severe symptoms with either virus within a shorter incubation period than did plants which were inoculated when their primary leaves were less than or greater than 50-75% expansion. The SMV strain, SMV-VA (G1/VA), was used to determine the cell types SMV moves into, using pinwheel inclusions as indicators of cell infection, at an early stage of infection when virus particles were first detectable in inoculated and noninoculated tissues of prilutry leaves. By using serologically specific electron microscopy (SSEM), virus particles were first detected in inoculated tissues at 6 days after inoculation. Pinwheels were detected within these areas in palisade, paraveinal, spongy, vascular parenchyma, phloem and immature xylem cells. The pinwheels were frequently opposed to the plasmalemma and were associated with plasmodesmata and plasmalemmasomes. Endoplasmic reticulum was associated with the base of those pinwheels found in the cytoplasm. The early stage of SMV pinwheel formation appeared to be similar to that reported for other potyviruses. The observation of pinwheels in the paraveineal mesophyll cells suggested the involvement of these cells in virus translocation within the soybean leaf. A sinusoidal pattern of SMV particle concentration within the inoculated and noninoculated areas of the leaf was detected by SSEM. The appearance of necrotic lesions within the inoculated area was correlated with an increase in the number of detectable particles within the area. A similar correlation was made with an increase in particle numbers in the non-inoculated area and the appearance of vein-clearing on the first trifoliolate leaf. It was also noted that particle numbers increased in the midrib and petiole when particle numbers decreased in the inoculated and noninoculated areas. This pattern was hypothesized to be due to either an alteration in the viral replication rate or to translocation relationships within the leaf. / Ph. D.

A study was conducted for four years to determine the effect of three soils at each of three Virginia locations on the protein and oil content of different soybean varieties. Soybeans were grown in plastic lined "pots'' fashioned by digging holes 48 inches deep and 14 inches in diameter. Root growth was restricted to the "pots" by plastic liners on the sides and copper mesh wire covering the bottom of each hole. Each soil was placed in the holes as near to its original position as possible. Under the conditions of this study, both soil type and location affected the oil and protein content of soybeans. Soil type affected protein percentage more than location but oil percentage was affected more by locations. Varieties responded differentially to soil type and location. Soybeans grown on Bertie sandy clay loam contained a higher protein and lower oil percentage than when grown on Sassafras or Davison soils. Soybeans grown on Davidson soil were lower in protein percentage than when grown on either Sassafras or Bertie soil. Location affected protein percentage in two out of four years and oil percentage each year. Soybeans grown on the southern-most location produced more oil and less protein than when produced at the two northern-most locations. / Master of Science

Master of Science

Virginia's Coastal Plain region contains the majority of the state's agricultural production despite having low nutrient soils. The soils in this region are predominantly coarse-textured acid soils with low cation exchange capacities (CEC) (< 3 cmol kg-1) and thus frequently exhibit nutrient deficiencies, including cationic nutrients which are not easily lost by leaching in soils with greater CEC. As a result, soils require careful nutrient management to maintain production levels. Soybean (Glycine max), the world's fourth largest crop, shows sensitivity to manganese availability and regularly experiences deficiency symptoms in low-CEC coastal plain soils. Tomato (Solanum lycopersicum) production, one of the 3 largest vegetable production systems in the world, requires careful management of various nutrients, particularly phosphorous, sulfur, and boron, for proper fruit development. Two novel coated fertilizer products consisting of granular KCl coated in a nutrient powder and a sugar-acid chelating agent are investigated as multi-nutrient sources for soybeans and tomatoes. A comprehensive review of the chemistry, behavior, and functionality of key nutrients provided by the fertilizer (P, S, Mn, and B) in both soils and plant tissues and the current state of chelate use in agriculture is provided along with related production issues with tomatoes and soybean. A greenhouse study investigating the ability of the first coated product (Mn + B coated KCl) to provide micronutrients to soybeans was conducted. Using both low and high organic matter (OM) soils (10 g kg-1; 36 g kg-1), Mn + B coated KCl increased soil Mn compared to no fertilizer and uncoated KCl. Additionally, Mn + B coated KCl increased total above ground tissue Mn compared to control and uncoated KCl for the low OM soil but not for the high OM soil, which was likely due to OM leading to the formation of metal-ligand complexes. There were no significant results regarding B concentration in either the soil or plant tissue due to the low application rate provided by the coating. The same fertilizer (Mn + B coated KCl) was investigated under field conditions to determine if increased soil and tissue Mn can be maintained under various environmental factors. Our results found that for all growing seasons and locations, there were no significant treatment differences between months for both Mn and B, but total monthly averages did fluctuate between months, probably reflecting changes in soil moisture and redox status. When averaged across the entire growing season, differences between treatments were inconsistent. Under field conditions, environmental conditions such as soil moisture and leaching likely masked any consistent treatment effects of the coated products. Two potential soil amendments, P + S + B coated KCl and glucoheptonate (GH), were investigated for their ability to provide nutrients to tomatoes. Three greenhouse trials, each lasting 3 weeks, were conducted. In the first trial, P + S + B coated KCl was compared to the current agronomic recommendation rates for P, S, and B. The coated KCl significantly increased soil and plant tissue P and B compared to all but the KCl + P and KCl + B treatments. The second trial was a glucoheptonate rate trial and showed a significant positive correlation between GH rate and soil and tissue B. The third trial combined and compared the coated KCl and GH products and showed that the treatments containing the coated KCl had significantly increased P, S, and B soil and tissue concentrations, with GH application having no synergistic effect / Ph. D. / The majority of the Virginia’s agricultural production occurs on the nutrient poor soils of the coastal plains where nutrient deficiencies are common. As a result, careful nutrient management strategies are required to maintain crop production levels, including major crops like soybean and tomato. Soybean (Glycine max) show sensitivity to manganese (Mn) availability and regularly experience deficiency symptoms in this region. On the other hand, tomato (Solanum lycopersicum) production requires careful management of nutrients such as phosphorous, sulfur, and boron for proper fruit development. In this dissertation, two novel coated fertilizer products, granular KCl coated in a nutrient powder and a sugar-acid chelating agent, are investigated as multi-nutrient sources for soybeans and tomatoes. This dissertation starts with a comprehensive review of the chemistry, behavior, and functionality of key nutrients provided by the fertilizer in soils and plant tissues, followed by a review of the current state of sugar acid use in agriculture. The production systems for soybeans and tomatoes for VA and the USA will also be discussed. The second component of this dissertation is a greenhouse study investigating the ability of the first coated product (Mn+B coated KCl) to provide micronutrients to soybeans. The Mn+B coated KCl significantly increased Mn compared to control and uncoated KCl treatments in the soil for both soil types and in tissue for the low OM soil. The third component of this dissertation investigates the same fertilizer under field conditions. Our results showed that for all growing seasons and locations, there were no significant treatment differences between months for both Mn and B, but monthly averaged concentrations did fluctuate over time, probably reflecting seasonal environmental shifts. When averaged annually, inconsistent differences were seen between treatments. Under field conditions, environmental conditions like increased soil moisture and leaching likely masked any consistent treatment effects of the coated products. The fourth component of this dissertation investigates two potential soil amendments, the second coated KCl product (P+S+B coated KCl) and glucoheptonate (GH), for their ability to provide nutrients to tomatoes. The study consists of 3 separate greenhouse trials, each lasting 3 weeks. In the first trial, P+S+B coated KCl was compared to the current agronomic recommendation rates for P, S, and B. The coated KCl significantly increased soil and plant tissue P and B compared to all but the KCl + P and KCl + B treatments. The second trial was a glucoheptonate rate trial and showed a significant positive correlation between GH rate and soil and tissue B. The third trial combined and compared the coated KCl and GH products and showed that the treatments containing the coated KCl had significantly increased P, S, and B soil and tissue concentrations, with GH application having no enhancing effect.

Increased demand for barley has changed the proportion of crops grown in Virginia and the Mid-Atlantic USA. Winter wheat is the predominant small grain crop, but barley can be a direct substitute, although much less of it is grown. Soybean is grown full-season and double-cropped after both small grains. Historically, wheat was the primary small grain in the soybean double-crop rotation because of its greater profitability. The barley-soybean cropping system is not a new concept in the region, but the literature is outdated. New agronomic and economic data that directly compares full-season soybean, barley-soybean, and wheat-soybean systems using modern cultivars and management practices is needed. The objectives of this research were to: i) determine soybean yield and compare cropping system profitability of the three cropping systems; ii) perform a breakeven sensitivity analysis of the three cropping systems; and iii) determine the effect of planting date and previous winter crop on soybean yield and yield components. Soybean grown after barley yielded more than full-season soybean in two of six locations and more than soybean double-cropped after wheat in three of six locations. Net returns for the barley-soybean system were the greatest. These data indicate that soybean double-cropped after barley has the potential to yield equal to or greater than full-season soybean or double-cropped soybean following wheat, but its relative yield is very dependent on growing conditions. The profitability comparison indicated that the barley-soybean cropping system was generally more profitable than the full-season soybean and double-cropped wheat-soybean systems. This conclusion was supported by the breakeven sensitivity analysis, but remains dependent on prices that have been extremely volatile in recent years. In another study, soybean yields declined with planting date at two of four locations in 2009, a year that late-season rainfall enabled later-planted soybean to yield more than expected. In 2010, soybean yield decline was affected by the delay in planting date at both locations. Winter grain did not affect soybean yield in either year. Yield component data reinforced these results and indicated that the lower seed yield in the later planting dates was due primarily to a decrease in the number of pods. / Master of Science