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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 β-conglycinin and 11S glycinin were mainly responsible for the observed increase in the total amount of protein in experimental lines., (Published by Elsevier B.V.)

The methionine-rich seed storage proteins of maize have been expressed in transgenic plants as a means to improve the overall sulfur amino acid content of seed. Previous attempts to increase the sulfur amino acid content of soybean seeds by this approach has met with limited success. It has been shown co-expression of different class of zeins can result in their stable accumulation in transgenic plants. In this study, conventional crosses between transgenic plants individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein were made to obtain plants that simultaneously express both the δ-zein and γ-zein. Transmission electron microscopic observation of thin-sections of transgenic soybean seeds revealed that the zeins accumulated in ER-derived protein bodies (PBs) which were found sparsely scattered in cytoplasm. The size of these PBs varied from 0.2 to 0.6 μm in soybean plants individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein. In contrast, soybeans co-expressing the 18 kDa δ-zein and 27 kDa γ-zein the PBs was 3-4 times larger. Electron microscopic observation also revealed the sequestration of PBs inside the vacuoles where they could be subjected to degradation by vacuolar proteases. Amino acid analysis of transgenic soybean individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein revealed only a minimal increase in the overall methionine content compared to the wild-type. In contrast, plants co-expressing 18 kDa δ-zein and 27 kDa γ-zein showed a significant increase (27%) in the methionine content compared to the control seeds., (Published by Elsevier B.V.)

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., (Published by Elsevier B.V.)

Specific metabolic network responses to mineral deficiencies are not well-defined. Here, we conducted a detailed broad-scale identification of metabolic responses of tomato leaves and roots to N, P or K deficiency. Tomato plants were grown hydroponically under optimal (5mM N, 0.5mM P, or 5mM K) and deficient (0.5mM N, 0.05mM P, or 0.5mM K) conditions and metabolites were measured by LC-MS and GC-MS. Based on these results, deficiency of any of these three minerals affected energy production and amino acid metabolism. N deficiency generally led to decreased amino acids and organic acids, and increased soluble sugars. P deficiency resulted in increased amino acids and organic acids in roots, and decreased soluble sugars. K deficiency caused accumulation of soluble sugars and amino acids in roots, and decreased organic acids and amino acids in leaves. Notable metabolic pathway alterations included; (1) increased levels of α-ketoglutarate and raffinose family oligosaccharides in N, P or K-deficient tomato roots, and (2) increased putrescine in K-deficient roots. These findings provide new knowledge of metabolic changes in response to mineral deficiencies., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Kunitz trypsin inhibitor, an abundant soybean [Glycine max (L.) Merr.] seed protein, has a molecular mass of 21500 Da and is specific for serine proteases. A soybean mutant (P.I. 196168) was characterized to determine the molecular basis for reduced Kunitz trypsin inhibitor levels during seed development. Western blot analysis revealed that P.I. 196168, in comparison to Amsoy 71, accumulated low amounts of Kunitz trypsin inhibitor protein. Non-denaturing polyacrylamide enzyme activity gels indicated that Amsoy 71 seeds contained at least five distinct zones of trypsin inhibitor activity. However, P.I. 196168 contained only four zones of enzyme inhibition. The coding region of the most abundant trypsin inhibitor gene (KTi3) was isolated from Amsoy 71 and P.I. 196168 by PCR. DNA sequence comparisons of the Kunitz trypsin inhibitor coding regions revealed two deletions and one G to T transversion have occurred. These mutations introduced four stop codons in the reading frame, resulting in a truncated protein. Northern blot analysis revealed that P.I. 196168 accumulated drastically lower amounts of KTi3 mRNA when compared with Amsoy 71.

A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised against the gel-purified protein. N-terminal sequence analysis identified the 53 kDa protein as the beta-subunit of beta-conglycinin. Results from Western blot analysis, using antibodies raised against the purified beta-subunit of beta-conglycinin, revealed that accumulation of this protein was enhanced in non-nodulating soybeans when the plants were supplemented with nitrogen. Results from Northern blot analysis indicate that non-nodulating soybeans in general had lower levels of mRNA for the major soybean seed proteins. A one-time application of nitrogen to non-nodulating soybeans enhanced the accumulation of the 1.6 kb beta-conglycinin beta-subunit mRNA. The mRNA levels of the 2.1 kb beta-conglycinin alpha'-subunit and the 2.2 kb G4 glycinin in the non-nodulating soybeans were several-fold lower than in nodulating soybeans. Nitrogen application had no effect on the abundance of these RNA transcripts. The amount of RNA encoding an 8.5 kDa sulfur-rich protein was two-fold higher in non-nodulating soybeans when compared with nodulated soybeans. Nitrogen application reduced the abundance of this transcript to levels comparable with those of nodulated soybeans. Despite lower levels of the beta-subunit of beta-conglycinin, the methionine content of the total seed protein fraction was lower than that of nodulated soybeans. In contrast, non-nodulating soybeans contained more cysteine than nodulating soybeans.