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Occurrence of mRNA for storage protein in dry soybean seeds.

Poly(A)-containing RNA has been isolated from the cotyledons of soybean seeds by adsorption on a poly(U)-Sepharose column. Approximately 0.15% of the total soybean RNA applied bound to the column. The bound RNA (poly(A)-containing RNA) was shown to be mRNA by its ability to serve as template in a cell-free system derived from wheat germ. Poly(A)-containing RNA was polydisperse, migrating from approximately 50,000 to 700,000 daltons with a mean of 150,000 daltons in polyacrylamide gel electrophoresis. The size of the poly(A) portion of this RNA was in the range of 55 to 290 nucleotides. The adenylic acid content of the presumed poly(A) fragment was about 95%. The radioactive products of translation directed by the poly(A)-containing RNA in the wheat germ cell-free system were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation using antisera against beta-conglycinin and glycinin. The results of this investigation show that mRNAs for the subunit proteins of the major components of a soybean storage protein exist in the poly(A)-containing RNA preparation obtained from the cotyledons of dry soybean seeds.

Cell-Free System

Nutrient comparison of fresh and field-dried, green-seeded soybeans.

Nutrient composition and biologic utilization of cooked, dried, and ground meals prepared from fresh and field-dried, green-seeded edible soybeans were evaluated. On a dry-weight basis, nutrient content of the fresh and field-dried meals were comparable for protein, fat, calcium, phosphorus, magnesium, copper, and iron; fresh beans tended to have higher zinc content than the field-dried beans. Nutrient values for the green-seeded soybean meals were comparable to published values for full-fat soybean flour. Bioassay results indicated that protein efficiency ratios (PER) for rats fed casein were significantly better than those for the soybean-fed animals. Fresh, green-seeded soybean meal supported significantly better growth than did the field-dried, green-seeded soybean meal. Though significantly lower than that for the reference casein diet, the mean PER for fresh, green-seeded soybean meal was 90 per cent of that obtained with the reference casein. The nutrient analysis and protein bioassay data both indicate that green-seeded soybeans used as a vegetable item in the diet are a potentially significant food source of several important nutrients.

Animals

Preliminary crystallographic data for Bowman-Birk inhibitor from soybean seeds.

A well characterized soybean protease inhibitor, the Bowman-Birk inhibitor, has been crystallized at room temperature in the presence of polyethylene glycol 4000 by vapor diffusion against an ammonium sulfate solution containing 2-methyl-2,4-pentanediol. An x-ray diffraction study reveals that the inhibitor crystallizes in a hexagonal unit cell of symmetry P6122 (or P6522) and dimensions a = b = 91.36(2) A and c = 63.92(2) A. Each of the 12 asymmetric units contains 2 molecules of molecular weight 8000. The crystal, which diffracts barely to 3-A spacings, is fairly stable to x-irradiation and has a solvent content of approximately 52% by volume.

Crystallization

Determination of free monosaccharides and detection of sugar alcohols in mature soybean seeds.

Although the oligosaccharide contents of soybeans are well documented, the exact values of monosaccharide contents have not been reported. Elaborate methods of preparative paper chromatography together with gas chromatography established the following data for one variety, Kyushu No. 12. The air-dried cotyledon part (admixed with hypocotyls) contained 0.030% glucose and 0.039% fructose. The hull part contained 0.018% galactose, 0.028% glucose, 0.023% fructose, 0.005% arabinose, and 0.002% xylose. Gas chromatograms of trimethylsilated monosaccharide fractions revealed the existence of minute amounts of sorbitol, arabitol, xylitol, and mannitol in decreasing order (about 0.03% to 0.001% of whole seeds).

Arabinose

Mechanistic roles of GmSWEET10a/b and GmSUT1 in the oil-protein balance in soybean mature seeds at transcriptional and metabolic levels.

Previous investigations indicated that the soybean (Glycine max) SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER10a/b (GmSWEET10a/b) genes promote oil accumulation, while inhibiting protein accumulation in seeds. To clarify the mechanisms modulated by GmSWEET10a/b in mediating the oil and protein accumulations in soybean seeds, an integrated comparative multiomics was conducted using the double gmsweet10a,b mutant and wild-type (WT) embryos. Spatial metabolomic analysis revealed that gmsweet10a,b embryos were surrounded by a sugar-reduced seed coat and experienced a sugar-starvation state in embryonic tissues in vivo. The decreased sugar content in the gmsweet10a,b embryos reduced the availability of carbon skeletons required for oil synthesis and was associated with decreased expression levels of genes involved in sucrose metabolism, fatty acid biosynthesis, and triacylglycerol assembly. Meanwhile, the expression of genes encoding storage protein was induced in gmsweet10a,b embryos, when compared with WT. These changes resulted in decreased oil content and increased protein content in gmsweet10a,b embryos versus WT. In vitro sugar-starvation assay also supported the suppression of fatty acid biosynthesis and the enhanced storage protein accumulation in developmental embryo under sugar-starved conditions. Furthermore, the knockout of SUCROSE TRANSPORTER 1 (GmSUT1), which was upregulated in gmsweet10a,b embryos, significantly decreased the sugar level, resulting in lower oil content but higher protein content in gmsut1 embryos than WT ones. Our findings provided a mechanistic understanding of the modulation of sugar transport between seed coat to embryo by both GmSWEET10a/b and GmSUT1, which plays a pivotal role in balancing oil and protein accumulations in soybean mature seeds.

Seeds

Targeted expression of Glycine max isoflavone synthase enhances daidzein and genistein content in soybean.

Isoflavonoids are key secondary metabolites in leguminous plants that play essential roles in plant physiology and provide significant health benefits to humans. In the isoflavone biosynthetic pathway, isoflavone synthase (IFS) catalyzes the conversion of naringenin and liquiritigenin into the bioactive isoflavones genistein and daidzein. This study aimed to enhance genistein and daidzein accumulation in soybean seeds through genetic engineering. Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301 containing GmIFS under the control of a seed-specific promoter (Gmβ-conglycinin) was used to transform modified half-seed explants of soybean cv. JS335. Hygromycin-B-resistant plants were regenerated, hardened, and confirmed by histochemical GUS assay. Molecular analysis by PCR validated the presence of the GmIFS transgene, yielding a 700 bp amplicon. Biochemical analysis revealed that seeds of T₀ transgenic plants showed a 1.53-fold increase in total phenolic content and a 3.67-fold increase in flavonoid content compared to non-transformed controls. Antioxidant assays demonstrated significantly higher DPPH radical-scavenging activity and ferric-reducing antioxidant power (FRAP) in GmIFS-overexpressing plants. HPLC analysis further indicated that transgenic seeds accumulated, on average, 4.07-fold higher daidzein and 1.75-fold higher genistein levels relative to control plants. qRT-PCR analysis showed significantly elevated GmIFS expression in immature cotyledons, mature cotyledons, and seeds of transgenic plants. Overall, these results demonstrate that GmIFS overexpression effectively enhances isoflavone production in soybean seeds, highlighting the potential of metabolic engineering of biosynthetic pathway genes to improve nutritional quality.

Glycine max

Natural variation in GmSOP5 regulates seed oil and protein content during soybean domestication.

Seed oil content, protein content, and yield are agronomically important, correlated traits that determine the economic value of soybean (Glycine max). However, improving seed quality and yield simultaneously is challenging because gains in one breeding target often compromise the other, and the genetic basis of this trade-off is poorly understood. Here, we performed a genome-wide association study of 429 diverse soybean accessions and identified Seed Oil and Protein 5 (SOP5), which encodes a kinesin protein, as a key locus associated with seed oil and protein content. Knockout and overexpression experiments demonstrated that GmSOP5 positively affects seed oil content and 100-seed weight and negatively influences seed protein content. GmSOP5 is located in a selective sweep region, and the domestication-related GmSOP5H1 allele is nearly fixed in cultivated soybean, contributing to increased seed size, weight, and oil content and reduced protein content. Field trials demonstrated that neither loss-of-function GmSOP5-edited mutants, which have increased seed protein content, nor GmSOP5-overexpression lines, which have increased seed oil content, differed significantly in yield from wild-type plants, because changes in plant architecture were offset by changes in seed weight. Our results shed light on soybean domestication and suggest how pleiotropy can be harnessed in breeding to enhance seed quality without compromising yield.

GWAS

Dissecting the genetic basis underlying drought tolerance at different development stages in soybean.

INTRODUCTION: Soybean is an indispensable crop supplying protein and oil for humans and animals, and playing an essential role in global food security. Drought represses soybean seed germination, reducing biomass accumulation and even inhibiting yield. METHODS: In order to dissect the genetic components underlying soybean drought tolerance during different development stage, a natural population containing 140 accessions was employed to evaluate seven drought tolerance-related traits under water-welled and drought stress conditions. Subsequently, genome-wide association study (GWAS) was conducted based on 150K single nucleotide polymorphism (SNP) markers of "Zhongdouxin-1". And the drought tolerance coefficient of seven different traits were analyzed with seven GWAS models. RESULTS: A total of 1807 significant SNPs were detected across 20 chromosome, including 569 SNPs for germination stage, and 1242 SNPs for seedling stage. Of 569 SNPs identified in germination stage, 354 SNPs on chromosomes 2, 7, 13, 14, and 17 accounting for 62.21%. Among 1242 SNPs found in seedling stage, 869 SNPs on chromosomes 11, 14, 15, 17 and 18 accounting for 69.97%. Moreover, among 1807 significant SNPs, 163 SNPs exhibited pleiotropic effects, of which 23 were located in exon, 21 in intron, 12 in 5'UTR or 3'UTR and 11 in upstream or downstream. Furthermore, 249 stable SNPs were detected by more than four GWAS models. According to these stable SNPs, RNA expression levels and gene annotations, four causal genes (Glyma.02G080200, Glyma.11G056200, Glyma.12G188900, and Glyma.18G110200) conferring soybean drought tolerance were detected, which participated in ethylene stimulus response, water deprivation response, and proteolysis. DISCUSSION: Collectively, 249 stable SNPs, 163 pleiotropic SNPs and four candidate genes identified in present study provided promising molecular resources and reliable foundation for drought resistance improvement and marker-assisted selective breeding in soybean.

GWAS

A soybean trypsin inhibitor. Crystallization and x-ray crystallographic study.

Five trypsin and alpha-chymotrypsin inhibitors which have low molecular weights (ranging from 6800 to 8600) and are present in soybean seeds of the Tracy variety have been isolated and purified, and single crystals which give x-ray diffraction data beyond 3-A spacings have been obtained from one of them. The trypsin inhibitor crystallizes in a monoclinic unit cell of symmetry P2(1) and dimensions a = 25.919(7) A, b = 43.23(1) A, c = 19.905(5) A, and beta = 103.63(2) degrees. The assymmetric unit contains 1 molecule of molecular weight 6800. The crystal, which has been found to be unusually stable to x-radiation, has solvent content of approximately 26% by volume.

Crystallization

Role of lectins in plant--microorganism interactions. IV. Ultrastructural localization of soybean lectin binding sites of Rhizobium japonicum.

The binding of purified, ferritin-labeled soybean seed lectin to the cell surfaces of Rhizobium japonicum 31 lb 138 has been examined by whole mount, thin section, and freeze-etch electron microscopy. The ferritin-labeled lectin binds in a biochemically specific manner to the capsular material of this bacterium. The lectin does not bind to the outer membranes of the cells or to flagella. Labeled lectin binds to sites throughout the capsular structure, although the density of labeling is somewhat greater on the outer surface of the capsule. Some cells appear to be partially encapsulated. Preservation of the capsular material proved difficult, and methods for retaining most of the capsular material were developed.

Binding Sites

Multiplex FAD2/FATB Editing Generates Ultra-High-Oleic, Low-Saturate Soybean With Increased Seed Fatty Acid Content.

Oleic acid (18:1), saturated fatty acid (SFA), and polyunsaturated fatty acid (PUFA) levels are important traits for storage stability and edibility. In this study, we aimed to develop high-oleic soybean (Glycine max) by simultaneously targeting fatty acid desaturase 2 (FAD2) and fatty acyl-ACP thioesterase B (FATB) gene families using CRISPR/Cas9. Considering the paleopolyploid genome of soybean, multiple sgRNAs were designed to target GmFAD2-1, GmFAD2-2, and GmFATB genes expressed during seed development. Compared with targeting GmFAD2-1 alone (~83%), additional editing of GmFAD2-2B, GmFAD2-2C, and GmFATB1a increased the 18:1 fatty acid content to over 90%. Editing of GmFATB significantly reduced the SFA content by more than 40% compared with wild-type (WT). Reduced absolute SFA content was also observed in GmFAD2-only edited lines with decreased GmFATB expression. Notably, all evaluated high-oleic genotypes in the Williams82-background showed higher mean total fatty acid (TFA) content than the WT, both per unit seed mass and per seed. Correlation analysis of 805 individual seed profiles from Williams82-background lines further characterized the relationship between 18:1 fatty acid composition and TFA content per unit seed mass. Gene expression analysis revealed no consistent increase in triacylglycerol (TAG) synthesis genes, whereas the expression of sugar-dependent 1 (SDP1) was reduced. In contrast, genes involved in phosphatidic acid (PA) metabolism, including diacylglycerol kinase (DGK) and phospholipase D (PLD), were partially upregulated. These transcriptional changes support a hypothesis that altered lipid turnover may contribute to the higher TFA phenotype. Collectively, this study defines effective multiplex target combinations for ultra-high oleic, low-saturated soybean.

FAD2

A wild soybean MADS-box gene GsAGL62 improves seed weight by enhancing cytokinin signaling and cell proliferation.

Soybean seed weight is a key yield determinant, but the transcriptional mechanisms connecting hormone signaling to seed growth are poorly understood. Here, we identify GsAGL62, a wild soybean MADS-box transcription factor located within a previously mapped hundred-seed weight (HSW) locus and a domestication-associated selective sweep. Functional analyses show that overexpression of GsAGL62 in cultivated soybean significantly increases HSW, whereas ethyl methanesulfonate (EMS)-induced gmagl62 mutants reduce it. Integrated transcriptomic and metabolomic analyses reveal that GsAGL62 enhances cytokinin accumulation and signaling cytokinin-associated responses, accompanied by increased expression of genes involved in cell proliferation. Mechanistically, GsAGL62 directly binds to the promoter of the conserved growth inhibitor GmATPK2 and represses its transcription. Consistently, independent EMS-induced gmatpk2 mutants exhibit increased seed weight, supporting GmATPK2 as a downstream negative regulator of seed growth. Population genetic analyses further reveal strong differentiation of GsAGL62 promoter haplotypes during soybean domestication and improvement. These haplotypes show differential promoter activities and are associated with distinct agronomic performance, suggesting that cis-regulatory variation at GsAGL62 contributes to its selection during soybean improvement. Collectively, our findings establish a regulatory module linking GsAGL62 to cytokinin-associated responses, cell proliferation, and seed growth, and highlight GsAGL62 as a potential target for soybean yield improvement.

Cell proliferation

Photoperiod and UV-B orchestrate ICR1 to control seed isoflavonoid accumulation in soybean.

Being sessile, plants optimize their physiological and metabolic processes in response to ambient environmental cues, such as light. However, the molecular mechanisms underlying environmental regulation of seed isoflavonoid biosynthesis in soybean remain largely elusive. Here, our genome-wide association study (GWAS) identifies Isoflavone Content Regulator 1 (ICR1), encoding a Regulator of Chromosome Condensation (RCC1) family protein, as a positive regulator of seed isoflavonoid accumulation in soybean. The CONSTANS (CO) homolog GmCOL2b directly suppresses ICR1 transcription by binding to a CORE cis-element. Notably, nature variations flanking the CORE sequence influence GmCOL2b binding affinity, thereby modulating ICR1 transcription and seed isoflavone content. We further demonstrate that photoperiod significantly affects seed isoflavone content, with short-day (SD) conditions promoting isoflavonoid accumulation by relieving GmCOL2b-mediated repression of ICR1, while UV-B radiation facilitates ICR1 protein accumulation. Subsequently, ICR1 interacts with GmMYB12B2 to enhance seed isoflavonoid biosynthesis. Our findings elucidate how soybean integrates environmental light signals, including photoperiod and UV-B signaling that vary across seasons or latitudes, to coordinate seed isoflavonoid biosynthesis, providing a valuable genetic resource for improving soybean nutritional quality.

Journal Article

GWAS-based identification of a candidate gene and development of a predictive KASP marker for seed protein and oil contents in soybean.

BACKGROUND: Soybean [Glycine max (L.) Merrill] is one of the most widely cultivated crops worldwide. Its seeds contain about 40% protein and 20% oil, serving as essential nutrient sources for humans. Given the nutritional importance of seed protein and oil, identifying genes that regulate their levels is crucial for improving soybean seed quality. OBJECTIVE: This study aimed to identify genetic factors associated with seed protein and oil content using a genome-wide association study (GWAS). METHODS: Seed protein and oil contents were quantified in 192 soybean mutant accessions in a mutant diversity pool (MDP), and GWAS was conducted using 17,631 SNPs filtered from genotyping-by-sequencing. Expression of a candidate gene was examined across seed developmental stages (R5 to R7), and a significant SNP was converted into a Kompetitive Allele-Specific PCR (KASP) marker for validation. RESULTS: GWAS detected significant SNPs associated with seed protein and oil content. Chr20_7635098 was identified as a nonsynonymous SNP located in the exon of Glyma.20g042400. This gene showed differential expression across seed developmental stages between mutant accessions with contrasting protein and oil contents. The KASP marker for Chr20_7635098 was validated using the MDP and six domestic soybean cultivars showing predictive accuracies of ≥ 80.50% for protein content and ≥ 61.18% for oil content. CONCLUSION: Overall, this study identified a candidate gene linked to both seed protein and oil content, providing valuable insights for molecular breeding strategies aimed at efficiently improving these nutritional traits.

Glycine max