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.