Elemental sulfur concentration can be used as a rapid, reliable, and cost-effective predictor of sulfur amino acid content of soybean seeds.

In this study, we have examined the feasibility of using elemental sulfur content of soybean seeds as a proxy for the overall sulfur amino acid content of soybean seeds. Earlier, we have identified by high throughput ionomic phenotyping several high and low sulfur containing soybean lines from the USDA Soybean Germplasm Collection. Here, we measured the cysteine and methionine content of select soybean lines by high-performance liquid chromatography. Our results demonstrate that those soybean lines which had high elemental sulfur content also had a higher cysteine and methionine content when compared to soybean lines with low elemental sulfur. SDS-PAGE and immunoblot analysis revealed that the accumulation of Bowman Birk protease inhibitor and lunasin in soybean seeds may only be marginally correlated with the elemental sulfur levels. However, we found a positive correlation between the levels of trypsin and chymotrypsin inhibitor activities and elemental sulfur and sulfur amino acid content of the seeds. Thus, elemental sulfur content and/or protease inhibitor activity measurement can be utilized as a rapid and cost-effective method to predict the overall sulfur amino acid content of soybean seeds. Our findings will benefit breeders in their endeavors to develop soybean cultivars with enhanced sulfur amino acid content.

Metabolic Profiling of a Fast Neutron Soybean Mutant Reveals an Increased Abundance of Isoflavones.

A total of 718 metabolites were identified in leaves and seeds of the soybean (Glycine max (L.) Merr., Fabaceae) fast neutron (FN) mutant 2012CM7F040p05ar154bMN15, which was previously shown to have 21 genes deleted and higher protein content in seeds as compared to wild-type. Among the identified metabolites, 164 were found only in seeds, 89 only in leaves, and 465 in both leaves and seeds. Metabolites that exhibited higher abundance in the mutant leaf than in the wild type include the flavonoids afromosin, biochanin A, dihydrodaidzein, and apigenin. Mutant leaves also exhibited a higher accumulation of glycitein-glucoside, dihydrokaempferol, and pipecolate. The seed-only metabolites that were found in higher abundance in the mutant compared to the wild type included 3-hydroxybenzoate, 3-aminoisobutyrate, coenzyme A, N-acetyl-ß-alanine, and 1-methylhistidine. Among several amino acids, the cysteine content increased in the mutant leaf and seed when compared to the wild type. We anticipate that the deletion of acetyl-CoA synthase created a negative feedback effect on carbon dynamics, resulting in increased amounts of cysteine and isoflavone-associated metabolites. Metabolic profiling provided new insight into the cascading effect of gene deletions that helps breeders to produce value-added nutritional seed traits.

Protein profiling of fast neutron soybean mutant seeds reveals differential accumulation of seed and iron storage proteins.

A fast neutron (FN) radiated mutant soybean (Glycine max (L.) Merr., Fabaceae) displaying large duplications exhibited an increase in total seed protein content. A tandem mass tag (TMT) based protein profiling of matured seeds resulted in the identification of 4338 proteins. Gene duplication resulted in a significant increase in several seed storage proteins and protease inhibitors. Among the storage proteins, basic 7 S globulin, glycinin G4, and beta-conglycinin showed higher abundance in matured FN mutant seeds in addition to protease inhibitors. A significantly higher abundance of L-ascorbate peroxidases, acid phosphatases, and iron storage proteins was also observed. A higher amount of albumin, sucrose synthase, iron storage, and ascorbate family proteins in the mutant seeds was observed at the mid-stage of seed filling. We anticipate that the duplicated genes might have a cascading effect on the genome constituents, thus, resulting in increased storage and iron-containing protein content in the mutant seeds.

Development of soybean experimental lines with enhanced protein and sulfur amino acid content.

Soybean is the preferred protein source for both poultry and swine feed. However, this preferred status is being challenged due to competition from alternative feed ingredients. To overcome this, it becomes necessary for breeders to develop soybean cultivars that contain higher protein and better nutritional composition. In this study, we have developed experimental soybean lines that not only contain significantly higher amounts of protein but also improved sulfur amino acid content. This objective was achieved by crossing a O-acetylserine sulfhydrylase (OASS) overexpressing transgenic soybean line with elevated levels of sulfur amino acid content (CS) with a high protein Korean soybean cultivar (Lee 5). Introgression of high protein and overexpression of OASS was monitored in the experimental lines at each successive generation (F2-F6) by measuring protein content and OASS activity. The average protein content of transgenic CS and Lee 5 seeds were 34.8 % and 44.7 %, while in the experimental soybean lines the protein content ranged from 41.3 %-47.7 %, respectively. HPLC and inductively coupled plasma-mass spectrometry analyses revealed that all the experimental lines developed in this study contained significantly higher amounts of sulfur containing amino acids and elemental sulfur in the seeds. The sulfur amino acid (cysteine + methionine) content of the experimental lines ranged from 1.1 % to 1.26 % while the parents Lee 5 and CS had 0.79 % and 1.1 %, respectively. SDS-PAGE and western blot analysis demonstrated that the accumulation of Bowman-Birk protease inhibitor and lunasin, two sulfur amino acid rich peptides, were elevated in experimental soybean lines. High-resolution 2D-gel electrophoresis and Delta2D gel analysis validated that an overall increase in the different subunits of 7S ß-conglycinin and 11S glycinin were mainly responsible for the observed increase in the total amount of protein in experimental lines.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the ß-subunit of ß-conglycinin in soybean seeds.

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the ß-subunit of ß-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

BG-4 from Bitter Gourd (Momordica charantia) Differentially Affects Inflammation In Vitro and In Vivo.

BG-4 isolated from bitter gourd has been reported for anti-cancer properties. The objective was to evaluate the anti-inflammatory properties of BG-4 in vitro and in vivo. Comparative study of the anti-inflammatory properties of BG-4 in vitro and in vivo was conducted on lipopolysaccharide (LPS)-activated mouse macrophages, and on dextran sodium sulfate (DSS)-induced colitis in mice. BG-4 reduced the production of pro-inflammatory markers in LPS-activated macrophages. On the other hand, intraperitoneal administration of BG-4 in DSS-induced colitis led to colon shortening, elevated neutrophils infiltration and myeloperoxidase activity, presence of blood in the stool, and loss of body weight, with differential systemic and local effects on pro-inflammatory cytokines in vivo. The results demonstrated that BG-4 differentially affected inflammation in vitro and in vivo.

Review: The promise and limits for enhancing sulfur-containing amino acid content of soybean seed.

Soybeans are an excellent source of protein in monogastric diets and rations with ∼75% of soybeans produced worldwide used primarily for animal feed. Even though soybeans are protein-rich and have a well-balanced amino acid profile, the nutritive quality of this important crop could be further improved by elevating the concentrations of certain amino acids. The levels of the sulfur-containing amino acids cysteine and methionine in soybean seed proteins are inadequate for optimal growth and development of monogastric animals, which necessitates dietary supplementation. Subsequently, concerted efforts have been made to increase the concentrations of cysteine and methionine in soybean seeds by both classical breeding and genetic engineering; however, these efforts have met with only limited success. In this review, we discuss the strengths and weakness of different approaches in elevating the sulfur amino acid content of soybeans. Manipulation of enzymes involved in the sulfur assimilatory pathway appears to be a viable avenue for improving sulfur amino acid content. This approach requires a through biochemical characterization of sulfur assimilatory enzymes in soybean seeds. We highlight recent studies targeting key sulfur assimilatory enzymes and the manipulation of sulfur metabolism in transgenic soybeans to improve the nutritive value of soybean proteins.

BG-4, a novel bioactive peptide from momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages.

BACKGROUND: Bitter melon (Momordica charantia) is a commonly used food crop for management of a variety of diseases most notably for control of diabetes, a disease associated with aberrant inflammation. PURPOSE: To evaluate the anti-inflammatory property of BG-4, a novel bioactive peptide isolated from the seed of bitter melon. METHODS: Differentiated THP-1 human macrophages were pre-treated with BG-4 and stimulated with lipopolysaccharide. Pro-inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. The mechanism of action involving activation of NF-κB and phosphorylation of ERK and STAT3 was measured by western blot and immunofluorescence. The production of intracellular reactive oxygen species was evaluated by fluorescence microscopy and fluorescence spectrophotometry. RESULTS: BG-4 dose dependently reduce the production of pro-inflammatory cytokines IL-6 and TNF-α. The ability of BG-4 to reduce production of cytokines are associated with reduced phosphorylation of ERK and STAT3 accompanied by reduced nuclear translocation of p65 NF-κB subunit. The mechanism of action is reduction of LPS-induced production of intracellular reactive oxygen species. CONCLUSION: Our results demonstrated the ability of BG-4, a novel peptide from the seed of bitter melon, to exert anti-inflammatory action. This could explain the traditional use of bitter melon against diseases associated with aberrant and uncontrolled inflammation.

Impact of overexpression of cytosolic isoform of O-acetylserine sulfhydrylase on soybean nodulation and nodule metabolome.

Nitrogen-fixing nodules, which are also major sites of sulfur assimilation, contribute significantly to the sulfur needs of whole soybean plants. Nodules are the predominant sites for cysteine accumulation and the activity of O-acetylserine(thiol)lyase (OASS) is central to the sulfur assimilation process in plants. Here, we examined the impact of overexpressing OASS on soybean nodulation and nodule metabolome. Overexpression of OASS did not affect the nodule number, but negatively impacted plant growth. HPLC measurement of antioxidant metabolites demonstrated that levels of cysteine, glutathione, and homoglutathione nearly doubled in OASS overexpressing nodules when compared to control nodules. Metabolite profiling by LC-MS and GC-MS demonstrated that several metabolites related to serine, aspartate, glutamate, and branched-chain amino acid pathways were significantly elevated in OASS overexpressing nodules. Striking differences were also observed in the flavonoid levels between the OASS overexpressing and control soybean nodules. Our results suggest that OASS overexpressing plants compensate for the increase in carbon requirement for sulfur assimilation by reducing the biosynthesis of some amino acids, and by replenishing the TCA cycle through fatty acid hydrolysis. These data may indicate that in OASS overexpressing soybean nodules there is a moderate decease in the supply of energy metabolites to the nodule, which is then compensated by the degradation of cellular components to meet the needs of the nodule energy metabolism.

Metabolomics Approach To Understand Mechanisms of ß-N-Oxalyl-l-α,ß-diaminopropionic Acid (ß-ODAP) Biosynthesis in Grass Pea (Lathyrus sativus L.).

A study was performed to identify metabolic processes associated with ß-ODAP synthesis in grass pea using a metabolomics approach. GC-MS metabolomics was performed on seedlings at 2, 6, and 25 days after sowing. A total of 141 metabolites were detected among the three time points representing much of grass pea primary metabolism, including amino acids, carbohydrates, purines, and others. Principal component analysis revealed unique metabolite profiles of grass pea tissues among the three time points. Fold change, hierarchical clustering, and orthogonal projections to latent structures-discriminant analyses, and biochemical pathway ontologies were used to characterize covariance of metabolites with ß-ODAP content. The data indicates that alanine and nitrogen metabolism, cysteine and sulfur metabolism, and purine, pyrimidine, and pyridine metabolism were associated with ß-ODAP metabolism. Our results reveal the metabolite profiles in grass pea development and provide insights into mechanisms of ß-ODAP accumulation and degradation.

ß-N-Oxalyl-l-α,ß-diaminopropionic Acid (ß-ODAP) Content in Lathyrus sativus: The Integration of Nitrogen and Sulfur Metabolism through ß-Cyanoalanine Synthase.

Grass pea (Lathyrus sativus L.) is an important legume crop grown mainly in South Asia and Sub-Saharan Africa. This underutilized legume can withstand harsh environmental conditions including drought and flooding. During drought-induced famines, this protein-rich legume serves as a food source for poor farmers when other crops fail under harsh environmental conditions; however, its use is limited because of the presence of an endogenous neurotoxic nonprotein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP). Long-term consumption of Lathyrus and ß-ODAP is linked to lathyrism, which is a degenerative motor neuron syndrome. Pharmacological studies indicate that nutritional deficiencies in methionine and cysteine may aggravate the neurotoxicity of ß-ODAP. The biosynthetic pathway leading to the production of ß-ODAP is poorly understood, but is linked to sulfur metabolism. To date, only a limited number of studies have been conducted in grass pea on the sulfur assimilatory enzymes and how these enzymes regulate the biosynthesis of ß-ODAP. Here, we review the current knowledge on the role of sulfur metabolism in grass pea and its contribution to ß-ODAP biosynthesis. Unraveling the fundamental steps and regulation of ß-ODAP biosynthesis in grass pea will be vital for the development of improved varieties of this underutilized legume.

BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells.

Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity of BG-4 may be responsible for its capability to cause cytotoxicity to HCT-116 and HT-29 human colon cancer cells with ED50 values of 134.4 and 217.0 µg/mL after 48 h of treatment, respectively. The mechanism involved in the cytotoxic effect may be associated with induction of apoptosis as evidenced by increased percentage of HCT-116 and HT-29 colon cancer cells undergoing apoptosis from 5.4% (untreated) to 24.8% (BG-4 treated, 125 µg/mL for 16 h) and 8.5% (untreated) to 31.9% (BG-4 treated, 125 µg/mL for 16 h), respectively. The molecular mechanistic explanation in the apoptosis inducing property of BG-4 is due to reduced expression of Bcl-2 and increased expression of Bax leading to increased expression of caspase-3 and affecting the expression of cell cycle proteins p21 and CDK2. This is the first report on the anti-cancer potential of a novel bioactive peptide isolated from Momordica charantia in vitro supporting the potential therapeutic property of BG-4 against colon cancer that must be addressed using in vivo models of colon carcinogenesis.

IL-3 Decreases Cartilage Degeneration by Downregulating Matrix Metalloproteinases and Reduces Joint Destruction in Osteoarthritic Mice.

Osteoarthritis (OA) is a chronic disease of articular joints that leads to degeneration of both cartilage and subchondral bone. These degenerative changes are further aggravated by proinflammatory cytokines including IL-1ß and TNF-α. Previously, we have reported that IL-3, a cytokine secreted by activated T cells, protects cartilage and bone damage in murine models of inflammatory and rheumatoid arthritis. However, how IL-3 protects cartilage degeneration is not yet known. In this study, we investigated the role of IL-3 on cartilage degeneration under both in vitro and in vivo conditions. We found that both mouse and human chondrocytes show strong expression of IL-3R at gene and protein levels. IL-3 increases the expression of mouse chondrocyte-specific genes, Sox9 and collagen type IIa, which were downregulated by IL-1ß. Moreover, IL-3 downregulated IL-1ß- and TNF-α-induced expression of matrix metalloproteinases in both mouse and human chondrocytes. Interestingly, IL-3 reduces the degeneration of articular cartilage and subchondral bone microarchitecture in a mouse model of human OA. Moreover, IL-3 showed the preventive and therapeutic effects on cartilage degeneration induced by IL-1ß in micromass pellet cultures of human mesenchymal stem cells. Thus, to our knowledge, we provide the first evidence that IL-3 has therapeutic potential in amelioration of degeneration of articular cartilage and subchondral bone microarchitecture associated with OA.

Immunological Investigation for the Presence of Lunasin, a Chemopreventive Soybean Peptide, in the Seeds of Diverse Plants.

Lunasin, a 44 amino acid soybean bioactive peptide, exhibits anticancer and anti-inflammatory properties. All soybean varieties that have been examined contain lunasin. It has also been reported in a few other plant species including amaranth, black nightshade, wheat, barley, rye, and triticale. Interestingly, detailed searches of transcriptome and DNA sequence databases of cereals failed to identify lunasin-coding sequences, raising questions about the authenticity of lunasin in cereals. To clarify the presence or absence of lunasin in cereals and other plant species, an immunological investigation was conducted utilizing polyclonal antibodies raised against the first 20 amino acid N-terminal peptide (SKWQHQQDSCRKQLQGVNLT) and a 15 amino acid C-terminal peptide (CEKHIMEKIQGRGDD) of lunasin. Protein blot analyses revealed the presence of proteins from several plants that reacted against the lunasin N-terminal peptide antibodies. However, the same proteins failed to react against the lunasin C-terminal peptide antibodies. These results demonstrate that peptides identical to soybean lunasin are absent in seeds of diverse plants examined in this study.

Introgression of leginsulin, a cysteine-rich protein, and high-protein trait from an Asian soybean plant introduction genotype into a North American experimental soybean line.

Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur-containing amino acids could enhance the nutritive value of soybean meal. Leginsulin, a cysteine-rich peptide, predominantly accumulates in Asian soybean accessions but not in most North American cultivars. By screening diverse soybean accessions from the USDA Soybean Germplasm Collection, we were able to identify one plant introduction, PI 427138, as a high-protein line with relatively high amounts of both elemental sulfur and leginsulin. We introgressed these desirable traits from PI 427138 into an experimental line with the aim of improving the overall protein content and quality of seed proteins. Biochemical characterization of inbred progenies from the cross of LD00-3309 with PI 427138 grown at six locations revealed stable ingression of high protein, high elemental sulfur, and high leginsulin accumulation. Comparison of soybean seed proteins resolved by high-resolution 2-D gel electrophoresis in combination with Delta2D image analysis software revealed preferential accumulation of a few glycinin subunits contributed to the increased protein content in the introgressed lines. Amino acid analysis revealed that even though the leginsulin introgressed lines had higher protein, leginsulin, and elemental sulfur, the overall concentration of sulfur-containing amino acids was not significantly altered when compared with the parental lines. The experimental soybean lines developed during this study (Leg-3, Leg-7, and Leg-8) lack A5, A4, and B3 glycinin subunits and could be utilized in breeding programs to develop high-quality tofu cultivars.

An effective and simple procedure to isolate abundant quantities of biologically active chemopreventive Lunasin Protease Inhibitor Concentrate (LPIC) from soybean.

Lunasin is a 5-kDa soybean bioactive peptide with demonstrated anti-cancer and anti-inflammatory properties. Recently, purification methods have been developed to obtain gram quantities of lunasin. However, these methods are cumbersome, time consuming and cost-prohibitive. To overcome these constrains we have developed a novel method which involves extraction of soybean flour with 30% ethanol followed by preferential precipitation of lunasin and protease inhibitors by calcium. The calcium precipitated protein fraction, which we termed as Lunasin Protease Inhibitor Concentrate (LPIC), contains three abundant proteins with molecular weights of 21, 14 and 5 kDa. This simple procedure yields 3.2g of LPIC from 100g of soybean flour and the entire isolation procedure can be completed in less than 2h. Treatment of THP-1 human monocyte cell lines with LPIC resulted in suppression of lipopolysaccharide-stimulated cytokine expression, demonstrating that the LPIC isolated by our simple procedure is biologically active.

Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation.

Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5'-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3'-phosphoadenosine 5'-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway.

Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone.

Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75-3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His(169) and Asp(154) form a catalytic dyad for general base catalysis and that His(189) may stabilize the oxyanion reaction intermediate. Glu(177) helps to position Arg(203) and His(204) and the ß1c-ß2c loop for serine binding. A similar role for ionic interactions formed by Lys(230) is required for CoA binding. The GmSAT structures also identify Arg(253) as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.

Imbibition of soybean seeds in warm water results in the release of copious amounts of Bowman-Birk protease inhibitor, a putative anticarcinogenic agent.

Protease inhibitors play a protective role against pathogenic microorganisms and herbivorous insects. The two predominant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor (KTI) and Bowman-Birk protease inhibitor (BBI). In this study, we report that soybean seeds incubated in warm water release large amounts of proteins into the surrounding media. Two-dimensional gel electrophoresis analysis of the seed exudates resulted in the separation of 93 distinct protein spots out of which 90 spots were identified by LC-MS/MS. The basic 7S globulin and the BBI are the two predominant proteins found in the soybean seed exudates. In addition to 7S and 11S seed storage proteins, others known to protect the seeds against pathogens and pests including KTI, peroxidase, α-galactosidase, and endo-1.3-ß-glucanase were also identified in the seed exudates. Soybean seed exudate obtained by incubating the seeds in warm water was also able to inhibit the growth of human breast cancer cell line MCF-7. Since soybean seeds release large amounts of enzymatically active BBI when immersed in warm water, our procedure could be exploited as a simplified alternative method for the preparation of BBI concentrate which is being used as a cancer chemoprotective agent.

Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds.

Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.