Growth and transpiration of soybean genotypes with HaHB4® transcription factor for drought tolerance.

The expression of HaHB4® transcription factor reduces soybean sensitivity to abiotic stresses, such as water deficit. Studies that quantify the tolerance of HaHB4® soybean to the soil water content in comparison with cultivars currently sown in Brazil are lacking. The objective of this study was to determine the level of drought tolerance of soybean genotypes expressing the HaHB4® transcription factor (TS18-6-610108 and TS18-6-610084) and commercial cultivars (TMG 7063 IPRO and BS IRGA 1642 IPRO) subjected to water deficit during the vegetative phase. We used the fraction of transpirable soil water (FTSW) approach. Parameters related to leaf transpiration, dry matter accumulation, water use efficiency (WUE), and transpiration coefficient (TC) were evaluated in the four soybean genotypes and two treatments (T1-100% replacement of transpired water and T2-without replacement of transpired water). The FTSW threshold for the decline in transpiration was evaluated to identify the onset of water stress in soybean. TS18-6-610108 and BS IRGA 1642 IPRO maintains potential transpiration at low FTSW values. The TS18-6-610108 genotype has 14% higher WUE than the sensitive cultivar under water deficit. Under well-irrigated conditions, the HaHB4® genotypes showed the highest TC values, which indicate well-functioning physiological processes.

Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus.

TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored.

Gene expression during development and overexpression after Cercospora kikuchii and salicylic acid challenging indicate defensive roles of the soybean toxin.

KEY MESSAGE: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

The dynamic process of dietary soybean ß-conglycinin in digestion, absorption, and metabolism among different intestinal segments in grass carp (Ctenopharyngodon idella).

The present study aimed to investigate the dynamic process of soybean ß-conglycinin in digestion, absorption, and metabolism in the intestine of grass carp (Ctenopharyngodon idella). Fish fed with 80 g ß-conglycinin/kg diet for 7 weeks, the intestinal digestive enzyme was extracted to hydrolyze ß-conglycinin in vitro, the free amino acid and its metabolism product contents in intestinal segments were analyzed. The present study first found that ß-conglycinin cannot be thoroughly digested by fish intestine digestive enzyme and produces new products (about 60- and 55-kDa polypeptides). The indigestible ß-conglycinin further caused the free amino acid imbalance, especially caused free essential amino acid deficiency in the proximal intestine but excess in the distal intestine. Moreover, these results might be partly associated with the effect of ß-conglycinin in amino acid transporters and tight junction-regulated paracellular pathway. Finally, dietary ß-conglycinin increased the content of amino acid catabolism by-product ammonia while decreased the amino acid anabolism product carnosine content in the proximal intestine and distal intestine. Thus, the current study first and systemically explored the dynamic process of ß-conglycinin in digestion, absorption, and metabolism, which further supported our previous study that dietary ß-conglycinin suppressed fish growth and caused intestine injure.

Chungkookjang with High Contents of Poly-γ-Glutamic Acid Improves Insulin Sensitizing Activity in Adipocytes and Neuronal Cells.

We hypothesized that soybeans fermented with Bacillus spp. for 48 h (chungkookjang) would be rich in poly-γ-glutamate (γ-PGA) and would have greater efficacy for improving insulin sensitivity and insulin secretion in 3T3-L1 adipocytes, min6 cells, and PC12 neuronal cells. We screened 20 different strains of B. subtillus and B. amyloliquefaciens spp. for γ-polyglutamate (PGA) production and their anti-diabetic and anti-dementia activities in cell-based studies. Chungkookjang made with two B. amyloliquefaciens spp. (BA730 and BA731) were selected to increase the isoflavonoid and γ-PGA. Insulin-stimulated glucose uptake was higher in 3T3-L1 adipocytes given both chungkookjang extracts than in the cells given vehicle (control). The ethanol extract of BA731 (BA731-E) increased the uptake the most. Triglyceride accumulation decreased in BA731-E and BA731-W and the accumulation increased in BA730-W and BA730-E. The mRNA expression of fatty acid synthetase and acetyl CoA carboxylase was much lower in BA731-E and BA731-W and it was higher in BA730-W than the control. BA730-E and BA730-W also increased peroxisome proliferator-activated receptor (PPAR)-γ activity. Glucose-stimulated insulin secretion increased with the high dosage of BA730-W and BA730-E in insulinoma cells, compared to the control. Insulin contents and cell survival in high glucose media were higher in cells with both BA731-E and BA730-E. Triglyceride deposition and TNF-α mRNA expression were lower in BA731 than the control. The high-dosage treatment of BA730-E and BA731-E increased differentiated neuronal cell survival after treating amyloid-ß(25-35) compared to the control. Brain-derived neurotrophic factor and ciliary neurotrophic factor, indices of neuronal cell proliferation, were higher in BA730 and BA731 than in the control. Tau expression was also reduced in BA731 more than the control and it was a similar level of the normal-control. In conclusion, BA730 increased PPAR-γ activity and BA731 enhanced insulin sensitivity in the brain and periphery. BA730 and BA731 prevented and alleviated the symptoms of type 2 diabetes and Alzheimer's disease with different pathways.

Viscoelastic properties of soy protein isolate - pectin blends: Richer than those of a simple composite material.

Concentrated soy protein isolate (SPI) - pectin blends acquire fibrous textures by shear-induced structuring while heating. The objective of this study was to determine the viscoelastic properties of concentrated SPI-pectin blends under similar conditions as during shear-induced structuring, and after cooling. A closed cavity rheometer was used to measure these properties under these conditions. At 140 °C, SPI and pectin had both a lower G* than the blend of the two and also showed a different behavior in time. Hence, the viscoelastic properties of the blend are richer than those of a simple composite material with stable physical phase properties. In addition, the G'pectin was much lower compared with the G'SPI and G'SPI-pectin upon cooling, confirming that pectin formed a weak dispersed phase. The results can be explained by considering that the viscoelastic properties of the blend are influenced by thermal degradation of the pectin phase. This degradation leads to: i) release of galacturonic acid, ii) lowering of the pH, and iii) water redistribution from the SPI towards the pectin phase. The relative importance of those effects are evaluated.

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. BIOLOGICAL SIGNIFICANCE: The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from genome sequences, though there are over lapped proteins. Based on the demonstrated application of data stored in the database for functional analyses, it is suggested that these data will be useful for analyses of biological mechanisms in soybean. Furthermore, coupled with recent advances in information and communication technology, the usefulness of this database would increase in the analyses of biological mechanisms.

Expression and Functional Analysis of a Novel Group of Legume-specific WRKY and Exo70 Protein Variants from Soybean.

Legumes fix atmospheric nitrogen through symbiosis with microorganisms and contain special traits in nitrogen assimilation and associated processes. Recently, we have reported a novel WRKY-related protein (GmWRP1) and a new clade of Exo70 proteins (GmExo70J) from soybean with homologs found only in legumes. GmWRP1 and some of the GmExo70J proteins are localized to Golgi apparatus through a novel N-terminal transmembrane domain. Here, we report further analysis of expression and functions of the novel GmWRP1 and GmExo70J genes. Promoter-GUS analysis in Arabidopsis revealed distinct tissue-specific expression patterns of the GmExo70J genes not only in vegetative but also in reproductive organs including mature tissues, where expression of previously characterized Exo70 genes is usually absent. Furthermore, expression of some GmExo70J genes including GmExo70J1, GmExo70J6 and GmExo70J7 increases greatly in floral organ-supporting receptacles during the development and maturation of siliques, indicating a possible role in seed development. More importantly, suppression of GmWRP1, GmExo70J7, GmExo70J8 and GmExo70J9 expression in soybean using virus- or artificial microRNA-mediated gene silencing resulted in accelerated leaf senescence and reduced nodule formation. These results strongly suggest that legume-specific GmWRP1 and GmExo70J proteins play important roles not only in legume symbiosis but also in other processes critical for legume growth and development.

Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

Expression of wild soybean WRKY20 in Arabidopsis enhances drought tolerance and regulates ABA signalling.

The WRKY-type transcription factors are involved in plant development and stress responses, but how the regulation of stress tolerance is related to plant development is largely unknown. GsWRKY20 was initially identified as a stress response gene using large-scale Glycine soja microarrays. Quantitative reverse transcription-PCR (qRT-PCR) showed that the expression of this gene was induced by abscisic acid (ABA), salt, cold, and drought. Overexpression of GsWRKY20 in Arabidopsis resulted in a decreased sensitivity to ABA during seed germination and early seedling growth. However, compared with the wild type, GsWRKY20 overexpression lines were more sensitive to ABA in stomatal closure, and exhibited a greater tolerance to drought stress, a decreased water loss rate, and a decreased stomatal density. Moreover, microarray and qRT-PCR assays showed that GsWRKY20 mediated ABA signalling by promoting the expression of negative regulators of ABA signalling, such as AtWRKY40, ABI1, and ABI2, while repressing the expression of the positive regulators of ABA, for example ABI5, ABI4, and ABF4. Interestingly, GsWRKY20 also positively regulates the expression of a group of wax biosynthetic genes. Further, evidence is provided to support that GsWRKY20 overexpression lines have more epicuticular wax crystals and a much thicker cuticle, which contribute to less chlorophyll leaching compared with the wild type. Taken together, the findings reveal an important role for GsWRKY20 in enhancing drought tolerance and regulating ABA signalling.

The soybean GmbZIP1 transcription factor enhances multiple abiotic stress tolerances in transgenic plants.

Abscisic acid (ABA)-responsive element binding proteins (AREBs) are basic domain/leucine zipper transcription factors that bind to the ABA-responsive element (ABRE) in the promoter regions of ABA-inducible genes in plants. A novel bZIP transcription factor gene, GmbZIP1, encoding 438 amino acids with a conserved bZIP domain composed of 60 amino acids was isolated from salt-tolerant soybean cv. Tiefeng 8. Southern blotting showed that only one copy was present in the soybean genome. Phylogenetic analyses showed that GmbZIP1 belonged to the AREB subfamily of the bZIP family and was most closely related to AtABF2 and OsTRAB1. The expression of GmbZIP1 was highly induced by ABA, drought, high salt and low temperature; and GmbZIP1 was expressed in soybean roots, stems and leaves under different stress conditions. GmbZIP1 was localized inside the nuclei of transformed onion epidermal cells. Overexpression of GmbZIP1 enhanced the responses of transgenic plants to ABA and triggered stomatal closure under stresses, potentially leading to improved tolerances to several abiotic stresses such as high salt, low temperature and drought in transgenic plants. Furthermore, overexpression of GmbZIP1 affected the expression of some ABA or stress-related genes involved in regulating stomatal closure in Arabidopsis under ABA, drought and high salt stress conditions. A few AREB elements were detected in the promoter region of those ABA or stress-related genes, suggesting that GmbZIP1 regulates the ABA response or stomatal closure mediated by those downstream genes in transgenic Arabidopsis. Moreover, GmbZIP1 was used to improve the drought tolerance trait of Chinese wheat varieties BS93. Functional analysis showed that overexpression of GmbZIP1 enhanced the drought tolerance of transgenic wheat, and transcripts of GmbZIP1 were detected in transgenic wheat using RT-PCR. In addition, GmbZIP1 overexpression did not result in growth retardation in all transgenic plants, suggesting that GmbZIP1 may be a valuable genetic resource for engineering stress tolerance of crops.

Computational complementation: a modelling approach to study signalling mechanisms during legume autoregulation of nodulation.

Autoregulation of nodulation (AON) is a long-distance signalling regulatory system maintaining the balance of symbiotic nodulation in legume plants. However, the intricacy of internal signalling and absence of flux and biochemical data, are a bottleneck for investigation of AON. To address this, a new computational modelling approach called "Computational Complementation" has been developed. The main idea is to use functional-structural modelling to complement the deficiency of an empirical model of a loss-of-function (non-AON) mutant with hypothetical AON mechanisms. If computational complementation demonstrates a phenotype similar to the wild-type plant, the signalling hypothesis would be suggested as "reasonable". Our initial case for application of this approach was to test whether or not wild-type soybean cotyledons provide the shoot-derived inhibitor (SDI) to regulate nodule progression. We predicted by computational complementation that the cotyledon is part of the shoot in terms of AON and that it produces the SDI signal, a result that was confirmed by reciprocal epicotyl-and-hypocotyl grafting in a real-plant experiment. This application demonstrates the feasibility of computational complementation and shows its usefulness for applications where real-plant experimentation is either difficult or impossible.

Establishment of a protein reference map for soybean root hair cells.

Root hairs are single tubular cells formed from the differentiation of epidermal cells on roots. They are involved in water and nutrient uptake and represent the infection site on leguminous roots by rhizobia, soil bacteria that establish a nitrogen-fixing symbiosis. Root hairs develop by polar cell expansion or tip growth, a unique mode of plant growth shared only with pollen tubes. A more complete characterization of root hair cell biology will lead to a better understanding of tip growth, the rhizobial infection process, and also lead to improvements in plant water and nutrient uptake. We analyzed the proteome of isolated soybean (Glycine max) root hair cells using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and shotgun proteomics (1D-PAGE-liquid chromatography and multidimensional protein identification technology) approaches. Soybean was selected for this study due to its agronomic importance and its root size. The resulting soybean root hair proteome reference map identified 1,492 different proteins. 2D-PAGE followed by mass spectrometry identified 527 proteins from total cell contents. A complementary shotgun analysis identified 1,134 total proteins, including 443 proteins that were specific to the microsomal fraction. Only 169 proteins were identified by the 2D-PAGE and shotgun methods, which highlights the advantage of using both methods. The proteins identified are involved not only in basic cell metabolism but also in functions more specific to the single root hair cell, including water and nutrient uptake, vesicle trafficking, and hormone and secondary metabolism. The data presented provide useful insight into the metabolic activities of a single, differentiated plant cell type.

GmRAR1 and GmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean.

RAR1, SGT1, and HSP90 are important components of effector-triggered immunity (ETI) in diverse plants, where RAR1 and SGT1 are thought to serve as HSP90 co-chaperones. We show that ETI in soybean requires RAR1 and SGT1 but not HSP90. Rsv1-mediated extreme resistance to Soybean mosaic virus (SMV) and Rpg-1b-mediated resistance to Pseudomonas syringae were compromised in plants silenced for GmRAR1 and GmSGT1-2 but not GmHSP90. This suggests that RAR1- or SGT1-dependant signaling is not always associated with a dependence on HSP90. Unlike in Arabidopsis, SGT1 in soybean also mediates ETI against the bacterial pathogen P. syringae. Similar to Arabidopsis, soybean RAR1 and SGT1 proteins interact with each other and two related HSP90 proteins. Plants silenced for GmHSP90 genes or GmRAR1 exhibited altered morphology, suggesting that these proteins also contribute to developmental processes. Silencing GmRAR1 and GmSGT1-2 impaired resistance to virulent bacteria and systemic acquired resistance (SAR) in soybean as well. Because the Arabidopsis rar1 mutant also showed a defect in SAR, we conclude that RAR1 and SGT1 serve as a point of convergence for basal resistance, ETI, and SAR. We demonstrate that, although soybean defense signaling pathways recruit structurally conserved components, they have distinct requirements for specific proteins.

Ectopic expression of soybean GmKNT1 in Arabidopsis results in altered leaf morphology and flower identity.

Plant morphology is specified by leaves and flowers, and the shoot apical meristem (SAM) defines the architecture of plant leaves and flowers. Here, we reported the characterization of a soybean KNOX gene GmKNT1, which was highly homologous to Arabidopsis STM. The GmKNT1 was strongly expressed in roots, flowers and developing seeds. Its expression could be induced by IAA, ABA and JA, but inhibited by GA or cytokinin. Staining of the transgenic plants overexpressing GmKNT1-GUS fusion protein revealed that the GmKNT1 was mainly expressed at lobe region, SAM of young leaves, sepal and carpel, not in seed and mature leaves. Scanning electron microscopy (SEM) disclosed multiple changes in morphology of the epidermal cells and stigma. The transgenic Arabidopsis plants overexpressing the GmKNT1 showed small and lobed leaves, shortened internodes and small clustered inflorescence. The lobed leaves might result from the function of the meristems located at the boundary of the leaf. Compared with wild type plants, transgenic plants had higher expression of the SAM-related genes including the CUP, WUS, CUC1, KNAT2 and KNAT6. These results indicated that the GmKNT1 could affect multiple aspects of plant growth and development by regulation of downstream genes expression.

The cancer preventive peptide lunasin from wheat inhibits core histone acetylation.

Lunasin is a unique 43-amino acid cancer preventive peptide initially reported in soybean and barley and has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. We report here the core histone H3- and H-acetylation inhibitory properties of lunasin from wheat, a new source of the peptide and from the livers of rats fed with lunasin-enriched wheat (LEW) to measure bioavailability. A non-radioactive histone acetyl transferase assay was used to measure inhibition of core histone acetylation. The presence of lunasin in wheat was established by Western blot and identified by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). Lunasin isolated from wheat seeds at different stages of development inhibited core histone H3 and H4 acetylation in a dose-dependent manner. Lunasin extracted from liver of rats fed with lunasin-enriched wheat (LEW) also inhibited histone acetylation confirming that the peptide is intact and bioactive. The amounts of lunasin in the developing seeds and in the rat liver correlated extremely well with the extent of inhibition of core histone acetylation.

Nuclear receptors: potential biomarkers for assessing physiological functions of soy proteins and phytoestrogens.

Soy consumption is associated with decreased incidence of chronic diseases, including cardiovascular diseases, atherosclerosis, diabetes, osteoporosis, and certain types of cancers. However, consumption of high amounts of soy isoflavones may adversely influence endocrine functions, such as thyroid function and reproductive performance, because of their structural similarity to endogenous estrogens. Nuclear receptors are a group of transcription factors that play critical roles in the regulation of gene expression and physiological functions through direct interaction with target genes. Modulation of the abundance of these receptors, such as changing their gene expression, alters the sensitivity of the target cells or tissues to the stimulation of ligands, and eventually affects the relevant physiological functions, such as growth, development, osteogenesis, immune response, lipogenesis, reproductive process, and anticarcinogenesis. A number of studies have shown that the bioactive components in soy can modify the expression of these receptors in various tissues and cancer cells, which is believed to be a key intracellular mechanism by which soy components affect physiological functions. This review summarizes the current understanding of the modulation of nuclear receptors by soy proteins and isoflavones, and focuses especially on the receptors for estrogens, progesterone, androgen, vitamin D, retinoic acid, and thyroid hormones as well as the potential impact on physiological functions.

PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli.

To have knowledge of the effect of soybean PM2 protein in protecting dehydrated cells and its functional region, PM2 cDNA was isolated from soybean immature seeds. The recombinants expressing full-length PM2, truncated polypeptides of PM2A (aa 1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C (duplication of 22-mer repeating region) were constructed. By using SDS-PAGE and mass spectrometry approaches, these fusion polypeptides were identified and proved to be hydrophilic and heat-stable. Spot assays of BL/PM2 and BL/pET28 (as control) showed that protein PM2 increased salt tolerance (500 mM NaCl or 500 mM KCl) of Escherichia coli, rather than osmotic tolerance (1100 mM sorbitol). In addition, comparing the survival ratios of the transformants under 500 mM NaCl or 500 mM KCl stresses, the results showed that: (1) the survival ratios of BL/PM2 and BL/PM2B were quite similar, both showing much higher values than those of BL/pET28. (2) The survival ratios of BL/PM2C were much higher than those of BL/PM2, BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2 polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is an important functional region.

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability.

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.

Association of blood pressure with intake of soy products and other food groups in Japanese men and women.

BACKGROUND: Soy diet has been suggested to have antihypertensive effect in animal studies. The present study examined the cross-sectional relationship between blood pressure and intake of soy products and other food groups in Japanese men and women. METHODS: Blood pressure was measured in Japanese 294 men and 330 women (246 premenopausal and 84 peri- and postmenopausal women) who participated in a health check-up program provided by a general hospital. Intake of various food groups and nutrients was estimated from a validated semiquantitative food frequency questionnaire. RESULTS: In men, soy product intake was inversely significantly correlated with diastolic blood pressure (r = -0.12, P = 0.04) after controlling for age, total energy, smoking status, body mass index, and intake of alcohol, salt and seaweeds. The correlation of soy product intake with systolic blood pressure was of borderline significance (r = -0.10, P = 0.09). Systolic blood pressure was inversely correlated with intake of vegetables (r = -0.12, P = 0.04) and dairy products (r = -0.12, P = 0.05). There were no significant correlations between soy product intake and diastolic blood pressure in women. CONCLUSIONS: These results indicate a mild effect of soy intake on blood pressure reduction in men.