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Various species of rhizobium establish compatible symbiotic relationships with soybean ( Glycine max ) leading to the formation of nitrogen-fixing nodules in roots. The formation of functional nodules is mediated through complex developmental and transcriptional reprogramming that involves the activity of thousands of plant genes. However, host transcriptome that differentiate between functional or non-functional nodules remain largely unexplored. In this study, we investigated differential compatibilities between rhizobium strains ( Bradyrhizobium diazoefficiens USDA110 Bradyrhizobium sp. strain LVM105) and cultivated and wild soybeans. The nodulation assays revealed that both USDA110 and LVM105 strains effectively nodulate G. soja but only USDA110 can form symbiotic relationships with Williams 82. LVM105 formed pseudonodules on Williams 82 that consist of a central nodule-like mass that are devoid of any rhizobia. RNA-seq data revealed that USDA110 and LVM105 induce distinct transcriptome programing in functional mature nodules formed on G. soja roots, where genes involved in nucleosome assembly, DNA replication, regulation of cell cycle, and defense responses play key roles. Transcriptome comparison also suggested that activation of genes associated with cell wall biogenesis and organization and defense responses together with downregulation of genes involved in the biosynthesis of isoprenoids and antioxidant stress are associated with the formation of non-functional nodules on Williams 82 roots. Moreover, our analysis implies that increased activity of genes involved in oxygen binding, amino acid transport, and nitrate transport differentiates between fully-developed nodules in cultivated versus wild soybeans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zadegan, Kim, Abbas, Kim, Krishnan and Hewezi.)

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans- acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1 , and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1 -gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T 1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman-Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC , genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Identifying and developing ice recrystallization inhibitors from sustainable food proteins such as soy protein isolate (SPI) can lead to practical applications in both pharmaceutical and food industries. The objective of this study was to investigate the ice recrystallization inhibition (IRI) activity of SPI hydrolysates, and this was achieved by using an IRI activity-guided fractionation approach and relating IRI activity to interfacial molecular activity measured by vibrational sum frequency generation (VSFG). In addition, the impact of molecular weight (MW) and enzyme specificity was analyzed using three different proteases (Alcalase, trypsin, and pancreatin) and varying hydrolysis times. Using preparative chromatography, hydrolysates from each enzyme treatment were fractionated into five different MW fractions (F1-F5), which were then characterized by high-performance liquid chromatography (HPLC). All SPI hydrolysates had IRI activity, resulting in a 57-29% ice crystal diameter reduction when compared to native SPI. The F1 fraction (of 4-14 kDa) was most effective among all tested hydrolysates, while the lower MW peptide fractions lacked activity. One sample (SPI-ALC 20-F1) had a 52% reduction of ice crystal size at a lower concentration of 2% compared to the typical 4% used. SFG showed a difference in H-bonding and hydrophobic interactions of the molecules on the water/air interface, which may be linked to IRI activity. This study demonstrates for the first time the ability of SPI hydrolysates to inhibit ice crystal growth and the potential application of SFG to study molecular interaction at the interface that may help illustrate the mechanism of action.

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-β-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.

Gene co-expression network analysis is an efficient systems biology approach for the discovery of novel gene functions and trait-associated gene modules. To identify clusters of functionally related genes involved in soybean nodule formation and development, we performed a weighted gene co-expression network analysis. Two nodule-specific modules (NSM-1 and NSM-2, containing 304 and 203 genes, respectively) were identified. The NSM-1 gene promoters were significantly enriched in cis-binding elements for ERF, MYB, and C2H2-type zinc transcription factors, whereas NSM-2 gene promoters were enriched in cis-binding elements for TCP, bZIP, and bHLH transcription factors, suggesting a role of these regulatory factors in the transcriptional activation of nodule co-expressed genes. The co-expressed gene modules included genes with potential novel roles in nodulation, including those involved in xylem development, transmembrane transport, the ethylene signalling pathway, cytoskeleton organization, cytokinesis and regulation of the cell cycle, regulation of meristem initiation and growth, transcriptional regulation, DNA methylation, and histone modifications. Functional analysis of two co-expressed genes using TILLING mutants provided novel insight into the involvement of unsaturated fatty acid biosynthesis and folate metabolism in nodule formation and development. The identified gene co-expression modules provide valuable resources for further functional genomics studies to dissect the genetic basis of nodule formation and development in soybean., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Rapid and accurate measurement of trypsin inhibitor is critical for soy processors to assess the quality of soy meal. Currently, trypsin inhibitor activity is measured using the American Oil Chemists' Society (AOCS) and the American Association of Cereal Chemists International (AACCI) approved method. We have modified and improved the AACCI/AOCS approved method resulting in the elimination of several time-consuming steps and drastically reducing the assay volume. By employing our simplified procedure, we have measured trypsin inhibitor activity of several soy and soy products. A side-by side comparison of our simplified procedure with AOCS approved method revealed strikingly similar results indicating that several time-consuming and tedious steps associated with AACCI/AOCS approved methods can be eliminated without sacrificing the accuracy of the assay. Moreover, we demonstrate that our assay can also be carried out in 96-well microplates which will enable high-throughput screening of large number of soy meal samples., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The development of new biopesticides to control the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is urgent due to resistance evolution to various control methods. We tested an air-dried non-live preparation of Chromobacterium species Panama (Csp_P), against multiple corn rootworm species, including Bt-resistant and -susceptible WCR strains, northern (NCR, D. barberi Smith & Lawrence), and southern corn rootworm (SCR, D. undecimpunctata howardi Barber), in diet toxicity assays. Our results documented that Csp_P was toxic to all three corn rootworms species based on lethal (LC 50 ), effective (EC 50 ), and molt inhibition concentration (MIC 50 ). In general, toxicity of Csp_P was similar among all WCR strains and ~ 3-fold less toxic to NCR and SCR strains. Effective concentration (EC 50 ) was also similar among WCR and SCR strains, and 5-7-fold higher in NCR strains. Molt inhibition (MIC 50 ) was similar among all corn rootworm strains except NCR diapause strain that was 2.5-6-fold higher when compared to all other strains. There was no apparent cross-resistance between Csp_P and any of the currently available Bt proteins. Our results indicate that Csp_P formulation was effective at killing multiple corn rootworm strains including Bt-resistant WCR and could be developed as a potential new management tool for WCR control., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., (Published by Elsevier Ltd.)

Using high throughput tandem mass tag (TMT) based tagging technique, we identified 4172 proteins in three developmental stages: early, mid, and late seed filling. We mapped the identified proteins to metabolic pathways associated with seed filling. The elevated abundance of several kinases was observed from the early to mid-stages of seed filling, indicating that protein phosphorylation was a significant event during this period. The early to late seed filling stages were characterized by an increased abundance of proteins associated with the cell wall, oil, and vacuolar-related processes. Among the seed storage proteins, 7S (β-subunit) and 11S (Gy3, Gy4, Gy5) steadily increased in abundance during early to late stages of seed filling, whereas 2S albumin exhibited a decrease in abundance during the same period. An increased abundance of proteases, senescence-associated proteins, and oil synthesis proteins was observed from the mid to late seed filling stages. The mid to late stages of seed filling was also characterized by a lower abundance of transferases, transporters, Kunitz family trypsin, and protease inhibitors. Two enzymes associated with methionine synthesis exhibited lower abundance from early to late stages. This study unveiled several essential enzymes/proteins related to amino acid and protein synthesis and their accumulation during seed development. All data can be accessed through this link: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=38784ecbd0854bb3801afc0d89056f84. (Accession MSV000087577)., (© 2022 Wiley-VCH GmbH.)

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

Adenanthera pavonina , an underutilized tropical tree, is being promoted as an alternative food source for meeting the nutritional needs of human and animals. In this study, we have shown that trypsin inhibitors as one of the predominant proteins in the seeds of A. pavonina . DE-52 column chromatography resulted in the identification of four peaks with trypsin inhibitor activity. SDS-PAGE and immunoblot analyses revealed DE-52 peaks A and B were enriched in 17 and 15 kDa proteins and these proteins cross-reacted against soybean trypsin inhibitor antibodies. Simulated gastric fluid digestion revealed that the 15-17 kDa proteins are resistant to pepsin digestion. Roasting the seeds lowered the trypsin inhibitor activity while boiling intact seeds elevated the enzyme activity. However, the trypsin inhibitor activity was completely abolished when the seeds were boiled without their seed coats. Immunohistochemical detection and confocal microscopy demonstrated that trypsin inhibitors were localized in the cell cytosol., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The salt-soluble globulins, glycinins (11S globulin), and β-conglycinins (7S globulin), are the most abundant seed proteins of soybean seeds. Together, these two groups of proteins account for 60-70% of total soybean seed proteins. Proteomic assessment of the less abundant soybean seed proteins using general isolation protocols is challenging due to the overwhelming abundance of storage proteins. Development of a simple, fast, and inexpensive method to remove most storage proteins from a seed extract will significantly enhance the study of the nonabundant proteins within seeds. We have developed two simple methods for the depletion of abundant seed proteins resulting in the enrichment of low abundance proteins from soybean seeds. Here, we provide a detailed procedure for the isolation, separation, identification, and quantification of low abundance seed proteins of soybean., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The goal of our study was to design a simple and feasible method to obtain lunasin, a naturally-occurring bioactive peptide, from tofu whey wastewater. A combination of alcoholic precipitation of high-molecular weight proteins from the whey, isoelectric precipitation of lunasin enriched material, and purification via gel filtration chromatography was selected as the best approach using tofu whey prepared at the laboratory scale. This process was applied to tofu whey produced by a local tofu factory and 773 mg of 80% purity lunasin was obtained per kg of dry tofu whey. Significant reduction of nitric oxide, and pro-inflammatory cytokines TNF-α and IL-6 over lipopolysaccharide activated murine macrophages demonstrate its biological activity. Our three-step process is not only simpler and faster than the previously reported methods to obtain lunasin but provides a sustainable approach for the valorization of a waste product, promoting the better utilization of soybean nutrients and active compounds., (Published by Elsevier Ltd.)

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.)

DNA methylation has recently emerged as a powerful regulatory mechanism controlling the expression of key regulators of various developmental processes, including nodulation. However, the functional role of DNA methylation in regulating the expression of microRNA (miRNA) genes during the formation and development of nitrogen-fixing nodules remains largely unknown. In this study, we profiled DNA methylation patterns of miRNA genes during nodule formation, development, and early senescence stages in soybean ( Glycine max ) through the analysis of methylC-seq data. Absolute DNA methylation levels in the CG, CHH, and CHH sequence contexts over the promoter and primary transcript regions of miRNA genes were significantly higher in the nodules compared with the corresponding root tissues at these three distinct nodule developmental stages. We identified a total of 82 differentially methylated miRNAs in the nodules compared with roots. Differential DNA methylation of these 82 miRNAs was detected only in the promoter (69), primary transcript region (3), and both in the promoter and primary transcript regions (10). The large majority of these differentially methylated miRNAs were hypermethylated in nodules compared with the corresponding root tissues and were found mainly in the CHH context and showed stage-specific methylation patterns. Differentially methylated regions in the promoters of 25 miRNAs overlapped with transposable elements, a finding that may explain the vulnerability of miRNAs to DNA methylation changes during nodule development. Gene expression analysis of a set of promoter-differentially methylated miRNAs pointed to a negative association between DNA methylation and miRNA expression. Gene Ontology and pathways analyses indicate that changes in DNA methylation of miRNA genes are reprogrammed and contribute to nodule development through indirect regulation of genes involved in cellular processes and pathways with well-established roles in nodulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Piya, Lopes-Caitar, Kim, Pantalone, Krishnan and Hewezi.)

Mutagenesis through fast neutron (FN) radiation of soybean resulted in a mutant with a 15% increase in seed protein content. A comparative genomic hybridization analysis confirmed that the mutant is lacking 24 genes located at chromosomes 5 and 10. A tandem mass tag-based proteomic profiling of the wild type and the FN mutant revealed 3,502 proteins, of which 206 proteins exhibited increased abundance and 214 proteins showed decreased abundance. Among the abundant proteins, basic 7S globulin increased fourfold, followed by vacuolar-sorting receptor and protein transporters. The differentially expressed proteins were mapped on the global metabolic pathways. It was observed that there was an enrichment of 29 ribosomal proteins, 16 endoplasmic reticular proteins, and several proteins in export metabolic pathways. The deletion of the sequence-specific DNA binding transcription factor along with 23 other genes may have altered the negative regulation of protein syntheses processes, resulting in an increase in the overall protein content of the mutant seed. This mutant is a valuable resource for researchers to understand the metabolic pathways that may affect an increase in seed protein content (the mass spectrometry data files were submitted to massive.ucsd.edu # MassIVE MSV000084228).

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 β-subunit of β-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.

Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

High growth temperatures negatively affect soybean ( Glycine max (L.) Merr) yields and seed quality. Soybean plants, heat stressed during seed development, produce seed that exhibit wrinkling, discoloration, poor seed germination, and have an increased potential for incidence of pathogen infection and an overall decrease in economic value. Soybean breeders have identified a heat stress tolerant exotic landrace genotype, which has been used in traditional hybridization to generate experimental genotypes, with improved seed yield and heat tolerance. Here, we have investigated the seed protein composition and ultrastructure of cotyledonary parenchyma cells of soybean genotypes that are either susceptible or tolerant to high growth temperatures. Biochemical analyses of seed proteins isolated from heat-tolerant and heat-sensitive genotypes produced under 28/22 °C (control), 36/24 °C (moderate), and 42/26 °C (extreme) day/night temperatures revealed that the accumulation in soybean seeds of lipoxygenase, the β-subunit of β-conglycinin, sucrose binding protein and Bowman-Birk protease inhibitor were negatively impacted by extreme heat stress in both genotypes, but these effects were less pronounced in the heat-tolerant genotype. Western blot analysis showed elevated accumulation of heat shock proteins (HSP70 and HSP17.6) in both lines in response to elevated temperatures during seed fill. Transmission electron microscopy showed that heat stress caused dramatic structural changes in the storage parenchyma cells. Extreme heat stress disrupted the structure and the membrane integrity of protein storage vacuoles, organelles that accumulate seed storage proteins. The detachment of the plasma membrane from the cell wall (plasmolysis) was commonly observed in the cells of the sensitive line. In contrast, these structural changes were less pronounced in the tolerant genotype, even under extreme heat stress, cells, for the most part, retained their structural integrity. The results of our study demonstrate the contrasting effects of heat stress on the seed protein composition and ultrastructural alterations that contribute to the tolerant genotype's ability to tolerate high temperatures during seed development.

Spontaneous and radiation-induced mutants of soybean, despite loss of abundant seed proteins, have been reported to grow and reproduce normally without any apparent physiological abnormalities. Here, we report the development and characterization of a soybean line (BSH-2) that lacks several abundant seed storage proteins. One-dimensional and high-resolution two-dimensional gel electrophoresis revealed the absence of the α' and α subunits of β-conglycinin and G1, G2, G4, and G5 glycinin in the newly developed mutant line (BSH-2). Like our earlier developed soybean mutant line (BSH-3), the seeds of BSH-2 also accumulated high levels of free amino acids as compared with wild-type DN47 seeds. An examination of the germination rates revealed that both BSH-2 and BSH-3 had significantly lower germination rates compared with the parent line DN47. Two-dimensional gel electrophoresis analysis demonstrated that these mutants had slower rates of mobilization of seed storage proteins. The delayed mobilization of storage proteins in BSH-2 and BSH-3 seeds was also correlated with a delayed induction of proteolytic activity in the mutants when compared to DN47. Similarly, qRT-PCR analysis revealed distinct expression pattern of genes involved in proteolytic pathway in the mutants when compared to DN47. Transmission electron microscopy observation of soybean seeds at two germination stages revealed striking differences in the breakdown of protein storage vacuoles and lipid bodies in the mutants. Our study demonstrates that BSH-2 and BSH-3 are compromised in mobilization of storage reserves and the absence of abundant storage proteins may affect the seed germination efficiency and post-germinative seedling establishment., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Grass pea is an orphan legume that is grown in many places in the world. It is a high-protein, drought-tolerant legume that is capable of surviving extreme environmental challenges and can be a sole food source during famine. However, grass pea produces the neurotoxin β- N -oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a neurological disease. This crop is promising as a food source for both animals and humans if β-ODAP levels and other antinutritional factors such as protease inhibitors are lowered or removed. To understand more about these proteins, a proteomic analysis of grass pea was conducted using three different extraction methods to determine which was more efficient at isolating antinutritional factors. Seed proteins extracted with Tris-buffered saline (TBS), 30% ethanol, and 50% isopropanol were identified by mass spectrometry, resulting in the documentation of the most abundant proteins for each extraction method. Mass spectrometry spectral data and BLAST2GO analysis led to the identification of 1376 proteins from all extraction methods. The molecular function of the extracted proteins revealed distinctly different protein functional profiles. The majority of the TBS-extracted proteins were annotated with nutrient reservoir activity, while the isopropanol extraction yielded the highest percentage of endopeptidase proteinase inhibitors. Our results demonstrate that the 50% isopropanol extraction method was the most efficient at isolating antinutritional factors including protease inhibitors.

Grass pea ( Lathyrus sativus L.) is an important legume commonly grown in arid and semi-arid regions. This protein-rich legume performs well even under harsh environmental conditions and is considered to be a strategic famine food in developing countries. Unfortunately, its potential usage is greatly limited as a result of the presence of antinutritional factors, including the neuroexcitatory amino acid β- N -oxalyl-l-α,β-diaminopropionic acid (β-ODAP) and protease inhibitors. β-ODAP is responsible for a neurodegenerative syndrome that results in the paralysis of lower limbs, while protease inhibitors affect protein digestibility, resulting in reduced growth. Concerted research efforts have led to development of grass pea cultivars with reduced β-ODAP content. In contrast, very little information is available on the protease inhibitors of L. sativus . In this study, we have conducted biochemical characterization of 51 L. sativus accessions originating from different geographical regions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of seed globulins and prolamins revealed striking similarity in their protein profile, although geographic-specific variations in profiles was also evident. Measurement of Bowman-Birk chymotrypsin inhibitor (BBi) and Kunitz trypsin inhibitor (KTi) activities in accessions revealed striking differences among them. Amino acid sequence alignment of grass pea BBi and KTi revealed significant homology to protease inhibitors from several legumes. Real-time polymerase chain reaction analysis demonstrated high-level expression of BBi and KTi in dry seeds and weak expression in other organs. Our study demonstrates substantial variation in BBi and KTi among grass pea accessions that could be exploited in breeding programs for the development of grass pea lines that are devoid of these antinutritional factors.

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.

Sesbania herbacea , a native North American fast-growing legume, thrives in wet and waterlogged conditions. This legume enters into symbiotic association with rhizobia, resulting in the formation of nitrogen-fixing nodules on the roots. A flooding-induced anaerobic environment imposes a challenge for the survival of rhizobia and negatively impacts nodulation. Very little information is available on how S. herbacea is able to thrive and efficiently fix N 2 in flooded conditions. In this study, we found that Sesbania plants grown under flooded conditions were significantly taller, produced more biomass, and formed more nodules when compared to plants grown on dry land. Transmission electron microscopy of Sesbania nodules revealed bacteroids from flooded nodules contained prominent polyhydroxybutyrate crystals, which were absent in non-flooded nodules. Gas and ion chromatography mass spectrometry analysis of nodule metabolites revealed a marked decrease in asparagine and an increase in the levels of gamma aminobutyric acid in flooded nodules. 2-D gel electrophoresis of nodule bacteroid proteins revealed flooding-induced changes in their protein profiles. Several of the bacteroid proteins that were prominent in flooded nodules were identified by mass spectrometry to be members of the ABC transporter family. The activities of several key enzymes involved in nitrogen metabolism was altered in Sesbania flooded nodules. Aspartate aminotransferase (AspAT), an enzyme with a vital role in the assimilation of reduced nitrogen, was dramatically elevated in flooded nodules. The results of our study highlight the potential of S. herbacea as a green manure and sheds light on the morphological, structural, and biochemical adaptations that enable S. herbacea to thrive and efficiently fix N 2 in flooded conditions.

Here, we present the draft genome sequence of Bradyrhizobium sp. strain LVM 105, a soil bacterium that forms nitrogen-fixing nodules on the roots of partridge pea. The genome size was 8,386,213 bp, with a GC content of 63.8%. The draft assembly contained 97 scaffolds with an N 50 value of 415,026 bp., (Copyright © 2019 Krishnan et al.)

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with β-oxidation of α-linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value-added soybeans., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pigeon pea ( Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts- S. fredii USDA191 and B. diazoefficiens USDA110-and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix- and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens , due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.

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.)

Grass pea, a protein-rich, high-yielding, and drought-tolerant legume, is used as food and livestock feed in several tropical and subtropical regions of the world. The abundant seed proteins of grass pea are salt-soluble globulins, which can be separated into vicilins and legumins. In many other legumes, the members of vicilin seed proteins have been identified as major allergens. However, very little information is available on the allergens of grass pea. In this study, we have identified an abundant 47 kDa protein from grass pea, which was recognized by immunoglobulin E (IgE) antibodies from sera drawn from several peanut-allergic patients. The IgE-binding 47 kDa protein was partially purified by affinity chromatography on a Con-A sepharose column. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of the 47 kDa grass pea protein revealed sequence homology to 47 kDa vicilin from pea and Len c 1 from lentil. Interestingly the grass pea vicilin was found to be susceptible to pepsin digestion in vitro. We have also isolated a cDNA encoding the grass pea 47 kDa vicilin (β-lathyrin), and the deduced amino acid sequence revealed extensive homology to several known allergens, including those from peanut and soybean. A homology model structure of the grass pea β-lathyrin, generated using the X-ray crystal structure of the soybean β-conglycinin β subunit as a template, revealed potential IgE-binding epitopes located on the surface of the molecule. The similarity in the three-dimensional structure and the conservation of the antigenic epitopes on the molecular surface of vicilin allergens explains the IgE-binding cross-reactivity.

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.)

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., (Published by Elsevier GmbH.)

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.

A soybean experimental line (BSH-3) devoid of a subset of seed storage proteins was developed by crossing a mutant donor line "HS99B" with a Chinese cultivar "Dongnong47" (DN47). One-dimensional and high-resolution 2-D gel electrophoresis revealed the absence of G1 (A1 a B 2) , G2 (A 2 B1 a) , and G4 (A 5 A 4 B 3) glycinin and the α' subunit of β-conglycinin in BSH-3 seeds. Despite the lack of these abundant seed proteins, BSH-3 seeds still accumulated 38% protein. BSH-3 seeds also accumulated high levels of free amino acids as compared with DN47 seeds, particularly arginine, and the amount of several essential amino acids were significantly elevated in BSH-3 seeds. Elevated accumulation of α and β-subunit of β-conglycinin, G5 glycinin, Kunitz trypsin inhibitor, and Bowman-Birk protease inhibitor indicates seed proteome rebalancing in BSH-3 seeds. Immunoblot analysis using sera from soybean allergic patients demonstrated the complete lack of a major allergen (α' subunit of β-conglycinin) in BSH-3 seeds. However, elevated levels of other allergens were found in BSH-3 seeds due to proteome rebalancing. Transmission electron microscopy observation of mature seeds of BSH-3 revealed striking differences in the appearance of the protein storage vacuoles when compared with DN47.

The elemental content of a soybean seed is a determined by both genetic and environmental factors and is an important component of its nutritional value. The elemental content is chemically stable, making the samples stored in germplasm repositories an intriguing source of experimental material. To test the efficacy of using samples from germplasm banks for gene discovery, we analyzed the elemental profile of seeds from 1,653 lines in the USDA Soybean Germplasm Collection. We observed large differences in the elemental profiles based on where the lines were grown, which lead us to break up the genetic analysis into multiple small experiments. Despite these challenges, we were able to identify candidate single nucleotide polymorphisms (SNPs) controlling elemental accumulation as well as lines with extreme elemental accumulation phenotypes. Our results suggest that elemental analysis of germplasm samples can identify SNPs in linkage disequilibrium to genes, which can be leveraged to assist in crop improvement efforts.

A study was performed to identify metabolic processes associated with β-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 β-ODAP content. The data indicates that alanine and nitrogen metabolism, cysteine and sulfur metabolism, and purine, pyrimidine, and pyridine metabolism were associated with β-ODAP metabolism. Our results reveal the metabolite profiles in grass pea development and provide insights into mechanisms of β-ODAP accumulation and degradation.

During ongoing proteomic analysis of the soybean ( Glycine max (L.) Merr) germplasm collection, PI 603408 was identified as a landrace whose seeds lack accumulation of one of the major seed storage glycinin protein subunits. Whole genomic resequencing was used to identify a two-base deletion affecting glycinin 5 The newly discovered deletion was confirmed to be causative through immunological, genetic, and proteomic analysis, and no significant differences in total seed protein content were found to be due to the glycinin 5 loss-of-function mutation per se In addition to focused studies on this one specific glycinin subunit-encoding gene, a total of 1,858,185 nucleotide variants were identified, of which 39,344 were predicted to affect protein coding regions. In order to semiautomate analysis of a large number of soybean gene variants, a new SIFT 4G (Sorting Intolerant From Tolerated 4 Genomes) database was designed to predict the impact of nonsynonymous single nucleotide soybean gene variants, potentially enabling more rapid analysis of soybean resequencing data in the future., (Copyright © 2017 Gillman et al.)

Pigeonpea is one of the major sources of dietary protein for more than a billion people living in South Asia. This hardy legume is often grown in low-input and risk-prone marginal environments. Considerable research effort has been devoted by a global research consortium to develop genomic resources for the improvement of this legume crop. These efforts have resulted in the elucidation of the complete genome sequence of pigeonpea. Despite these developments, little is known about the seed proteome of this important crop. Here, we report the proteome of pigeonpea seed. To enable the isolation of maximum number of seed proteins, including those that are present in very low amounts, three different protein fractions were obtained by employing different extraction media. High-resolution two-dimensional (2-D) electrophoresis followed by MALDI-TOF-TOF-MS/MS analysis of these protein fractions resulted in the identification of 373 pigeonpea seed proteins. Consistent with the reported high degree of synteny between the pigeonpea and soybean genomes, a large number of pigeonpea seed proteins exhibited significant amino acid homology with soybean seed proteins. Our proteomic analysis identified a large number of stress-related proteins, presumably due to its adaptation to drought-prone environments. The availability of a pigeonpea seed proteome reference map should shed light on the roles of these identified proteins in various biological processes and facilitate the improvement of seed composition.

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 β- N -oxalyl-l-α,β-diaminopropionic acid (β-ODAP). Long-term consumption of Lathyrus and β-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 β-ODAP. The biosynthetic pathway leading to the production of β-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 β-ODAP. Here, we review the current knowledge on the role of sulfur metabolism in grass pea and its contribution to β-ODAP biosynthesis. Unraveling the fundamental steps and regulation of β-ODAP biosynthesis in grass pea will be vital for the development of improved varieties of this underutilized legume.

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8μg/g pre-hydrolysis and 171.5 to 250.1μg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3μg/g pre-hydrolysis and 9.0 to 18.7μg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0μg/g pre-hydrolysis and 4.0 to 13.2μg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Soy (Glycine max) is a major commodity in the United States, and soy foods are gaining popularity due to their reported health-promoting effects. In the past two decades, soy and soy bioactive components have been studied for their health-promoting/disease-preventing activities and potential mechanisms of action. Recent studies have identified gut microbiota as an important component in the human body ecosystem and possibly a critical modulator of human health. Soy foods' interaction with the gut microbiota may critically influence many aspects of human development, physiology, immunity, and nutrition at different stages of life. This review summarizes current knowledge on the effects of soy foods and soy components on gut microbiota population and composition. It was found, although results vary in different studies, in general, both animal and human studies have shown that consumption of soy foods can increase the levels of bifidobacteria and lactobacilli and alter the ratio between Firmicutes and Bacteroidetes. These changes in microbiota are consistent with reported reductions in pathogenic bacteria populations in the gut, thereby lowering the risk of diseases and leading to beneficial effects on human health.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria . Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234 a ). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades.

Perennial Glycine species, distant relatives of soybean, have been recognized as a potential source of new genetic diversity for soybean improvement. The subgenus Glycine includes around 30 perennial species, which are well-adapted to drought conditions and possess resistance to a number of soybean pathogens. In spite of the potential of the perennial Glycine species for soybean improvement, very little is known about their storage proteins and their relationship with cultivated soybean seed proteins. We have examined the seed protein composition of nine perennial Glycine species by one- and two-dimensional (1-D and 2-D) gel electrophoresis. The relationship between cultivated soybean and perennial soybean seed proteins was examined by immunoblot analyses using antibodies raised against G. max β-conglycinin, glycinin A3 subunit, lipoxygenase, leginsulin, Kunitz trypsin inhibitor, and Bowman-Birk protease inhibitor. Additionally, we have measured the trypsin and chymotrypsin inhibitor activities from cultivated soybean and perennial Glycine species and have found marked differences between them. Our 2-D gel and immunoblot analyses demonstrate significant differences in the protein composition and size heterogeneities of the 7S and 11S seed storage proteins of soybean and perennial Glycine species. Perennial Glycine species accumulated a 45 kDa protein that was not detected in G. max and G. soja. This unique 45 kDa protein was immunologically related to the A3 glycinin subunit of G. max. The results of our studies suggest that even though the seed proteins of wild perennial Glycine species and G. max are immunologically related, their genes have diverged from each other during the course of evolution.

Legumes form symbiotic associations with soil-dwelling bacteria collectively called rhizobia. This association results in the formation of nodules, unique plant-derived organs, within which the rhizobia are housed. Rhizobia-encoded nitrogenase facilitates the conversion of atmospheric nitrogen into ammonia, which is utilized by the plants for its growth and development. Fatty acids have been shown to play an important role in root nodule symbiosis. In this study, we have investigated the role of stearoyl-acyl carrier protein desaturase isoform C (SACPD-C), a soybean enzyme that catalyzes the conversion of stearic acid into oleic acid, which is expressed in developing seeds and in nitrogen-fixing nodules. In-depth cytological investigation of nodule development in sacpd-c mutant lines M25 and MM106 revealed gross anatomical alteration in the sacpd-c mutants. Transmission electron microscopy observations revealed ultrastructural alterations in the sacpd-c mutants that are typically associated with plant defense response to pathogens. In nodules of two sacpd-c mutants, the combined jasmonic acid (JA) species (JA and the isoleucine conjugate of JA) were found to be reduced and 12-oxophytodienoic acid (OPDA) levels were significantly higher relative to wild-type lines. Salicylic acid levels were not significantly different between genotypes, which is divergent from previous studies of sacpd mutant studies on vegetative tissues. Soybean nodule phytohormone profiles were very divergent from those of roots, and root profiles were found to be almost identical between mutant and wild-type genotypes. The activities of antioxidant enzymes, ascorbate peroxidase, and superoxide dismutase were also found to be higher in nodules of sacpd-c mutants. PR-1 gene expression was extremely elevated in M25 and MM106, while the expression of nitrogenase was significantly reduced in these sacpd-c mutants, compared with the parent 'Bay'. Two-dimensional gel electrophoresis and matrix-assisted laser desorption-ionization time of flight mass spectrometry analyses confirmed sacpd-c mutants also accumulated higher amounts of pathogenesis-related proteins in the nodules. Our study establishes a major role for SACPD-C activity as essential for proper maintenance of soybean nodule morphology and physiology and indicates that OPDA signaling is likely to be involved in attenuation of nodule biotic defense responses.

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.

Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max) near-isogenic line (cgy-2-NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. To identify α-null-related transcriptional changes, the gene expressions of cgy-2-NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). Seeds at 18 DAF served as the control. Comparison of the transcript profiles identified 3,543 differentially expressed genes (DEGs) between the two genotypes, with 2,193 genes downregulated and 1,350 genes upregulated. The largest numbers of DEGs were identified at 55 DAF. The DEGs identified at 25 DAF represented a unique pattern of GO category distributions. KEGG pathway analyses identified 541 altered metabolic pathways in cgy-2-NIL. At 18DAF, 12 DEGs were involved in arginine and proline metabolism. The cgy-2 allele in the homozygous form modified the expression of several Cupin allergen genes. The cgy-2 allele is an alteration of a functional allele that is closely related to soybean protein amino acid quality, and is useful for hypoallergenic soybean breeding programs that aim to improve seed protein quality.

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-1β 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-1β. Moreover, IL-3 downregulated IL-1β- 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-1β 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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

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.