Β-subunit Of Β-conglycinin Research Articles

The role of β-subunit in emulsifying performance of β-conglycinin

The most crucial protein that affects the ability of soybean protein to emulsify is β-conglycinin. The subunit composition of β-conglycinin is one of the important factors affecting its emulsification performance. In order to investigate the effect of β-subunit of β-conglycinin on its emulsifying property, the separated and purified β-subunit was added to β-conglycinin in different proportions, and the properties of the β-conglycinin before and after emulsification were measured. The β-subunit appeared to improve the stability of the β-conglycinin in the aqueous dispersion as evidenced by the average particle size significantly decreasing, the surface hydrophobicity significantly increasing, and the Emulsification activity index and Emulsion stability index further improving. The secondary structure of β-conglycinin was altered by the addition of β-subunit, which caused more conversion of β-sheet and β-turn to α-helix, increasing the flexibility of protein molecules. Additionally, the particle size distribution, ζ-potential and creaming index of the stable emulsion formed by β-conglycinin were also measured. The results showed that the increase of β-subunit content improved the stability of the emulsion to some extent. The stability of emulsion mainly relies on their internal structure to maintain. It could be concluded from rheological analysis that the viscosity of emulsion increases due to the presence of β-subunits and the existence of weak gel structure inside them, which is corroborated by the results of confocal laser scanning electron microscopy. The elucidation of the influence of subunits on the emulsification characteristics of soyabean protein is beneficial to the development of soybean protein industry.

Characteristics of molecular composition and its anti-nutrition of β-conglycinin during flavorzyme proteolysis

β-conglycinin is one of the main protein components in soybean that cause human allergy and it is difficult to eliminate the antigenicity of β-conglycinin using common enzymolysis, the reason of which is not clear. We analyzed the molecular composition, antigenic activity, and sequential epitopes of the hydrolysates of this protein after digestion with flavorzyme using SDS-PAGE, ELISA, MALDI-TOF-MS, and protein and antigen database retrieval. Results showed that α′-and α-subunits of β-conglycinin were nearly completely degraded 10 min after enzymolysis and that the protein retained 92.7% antigenic activity. Newly produced peptides, including those of 35, 23, 17, and 11 ku, were formed. These peptides and the 49 ku β-subunit were not degraded completely even after 150 min of enzymolysis, thus indicating evident resistance to enzymatic hydrolysis. Moreover, the antigenic activity of the protein maintained at 43.5%. Epitope analysis revealed that the newly formed 49, 35, 23, 17, and 11 ku peptides contain twenty seven antigen epitopes, including two from the α-subunit, ten from the α′-subunit, and fifteen from the β-subunit of β-conglycinin. These findings suggest the relationship between existence of anti-enzymolysis fragments and their epitopes and incomplete elimination of antigenicity of β-conglycinin.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman–Birk protease inhibitor and suppresses the accumulation of the β-subunit of β-conglycinin in soybean seeds

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.

Effect of Heat Stress on Seed Protein Composition and Ultrastructure of Protein Storage Vacuoles in the Cotyledonary Parenchyma Cells of Soybean Genotypes That Are Either Tolerant or Sensitive to Elevated Temperatures.

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.

Development and Characterization of a Soybean Experimental Line Lacking the α' Subunit of β-Conglycinin and G1, G2, and G4 Glycinin.

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 (A1aB2), G2 (A2B1a), and G4 (A5A4B3) 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.

Soybean seeds overexpressing asparaginase exhibit reduced nitrogen concentration

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K+ -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the β-subunit of β-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing β-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation.

Effect of Six Decades of Selective Breeding on Soybean Protein Composition and Quality: A Biochemical and Molecular Analysis

To evaluate the extent of the genetic change and its effects on the seed protein composition of soybean cultivars released during the past 60 years, representative ancestral cultivars and those derived from selective breeding were grown in a side-by-side comparison. Total seed protein content, determined by combustion analysis of nitrogen, revealed a decline in the protein content after decades of selection and breeding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis comparison of protein profiles of the soybean cultivars indicated that relative expression of most of the seed storage proteins had not varied substantially from the ancestral lines to the present commercial cultivars. There was noticeably less beta-subunit of beta-conglycinin, a protein devoid of sulfur amino acids, in the modern cultivars represented by Mustang, Pioneer 93B09, and Asgrow 3602. Comparison of the amino acid profiles of soybean seed, a benchmark of the protein's nutritional quality, revealed that the ancestral progenitor, G. soja, was significantly higher in cysteine, glutamic acid, histidine, and arginine than either the ancestral or the modern cultivars. Selective breeding over the past 60 years minimally affected the overall amino acid composition. The degree of divergence in the DNA sequence of the genes encoding glycinin and beta-conglycinin in the ancestral and modern cultivars was investigated using Southern hybridization and the polymerase chain reaction. Even though some restriction fragment polymorphisms could be detected, overall, the banding patterns were remarkably similar among the ancestral cultivars and those derived from them, suggesting a high degree of conservation of seed-storage protein genes. The results of our study suggest that selection and breeding for yield during the past 60 years had no major influence on the protein composition, ostensibly because of limited genetic diversity among the parental lines.

Evidence for accumulation of the β-subunit of β-conglycinin in soybean [Glycine max (L) Merr.] embryonic axes

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins from soybean cotyledons and embryonic axes revealed the presence of similar storage proteins in both organs. Results from Western blot analysis conducted with antibodies raised against the purified β-subunit of β-conglycinin demonstrated accumulation of the β-subunit in embryonic axes. This accumulation followed a temporal pattern similar to that shown by the cotyledons. Axis β-conglycinin was broken down during the initial stage(s) of seed germination and was completely mobilized within 3 days after imbibition. Subcellular fractions were isolated from developing embryonic axes using metrizamide density gradients and analyzed by Western blots. Storage proteins were enriched in the lighter fractions of the gradient as well as with immunoglobulin heavy-chain binding protein. Electron microscopy of the storage-protein-enriched gradient fraction revealed small vesicles and protein aggregates. Protein A-gold immunocytochemistry was used to localize the occurrence of the β-subunit of β-conglycinin within the protein aggregates present in the metrizamide gradient and in the protein bodies present in mature embryonic axes.

Effect of short-term application of nitrogen on the accumulation of β-subunit of β-conglycinin in nitrogen-starved soybean (Glycine max L.) developing seeds

Abstrct It has been reported that the accumulation of the β-subunit of β-conglycinin, a storage protein of soybean (Glycine max L.) seeds, was suppressed by nitrogen (N)-deficiency. In this report we attempted to determine which compound(s) may playa key role in regulating the accumulation of β-subunit mRNA and protein in intact seeds and in an in vitro cotyledon culture system. Non-nodulating soybean plants were cultivated under N-deficient (0.5 mm NO3 −) conditions, and transferred to N-sufficient medium (5 mm NO3 −) when the plants reached the pod filling stage. The β-subunit mRNA and the protein were detected in immature seeds within 2 d after transfer to 5 mm NO3 − medium. Among the free amino acids in the immature seeds, asparagine concentration increased rapidly within 2 d. In the in vitro cotyledon culture system, the application of glutamine induced the accumulation of β-subunit mRNA within 12 h, while asparagine did not induce the accumulation of β-subunit mRNA for 7 d. An inhibitor of transaminases, (aminooxy)acetic acid, enhanced β-subunit mRNA accumulation without Gln accumulation when asparagine was the sole nitrogen source. Although the asparagine concentration in the seed reflected the nitrogen statues in whole plant, the accumulation of β-subunit mRNA and continuous protein storage in immature seeds appeared to be regulated by glutamine, its metabolites or related compounds.

Temporary application of nitrate to nitrogen-deficient soybean plants at the mid- to late-stages of seed development increased the accumulation of the β-Subunit of β-conglycinin, a major seed storage protein

The accumulation of the β-subunit of β-conglycinin, a major soybean storage protein, in seeds is controlled by the nitrogen (N) status of the plant. When non-nodulating soybean mutants (T201) were hydroponically cultivated under N-sufficient conditions (supply of 5 mM NaN03 to the medium), β-subunit accumulation was first detected at 30 d after flowering (DAF). In N-deficient plants (supply of 1 mM NaNO3), the accumulation of the β-subunit was stronglyinhibited. When 5 mM 15N-labeled NO3 - was supplied for 1 week to N-deficient T201 plants before 28 DAF, the β-subunit protein did not accumulate, but the application of 5 mM NO3 - after 28 DAF, led to a substantial accumulation of the β-subunit. The total 15N percentage in mature seeds was similar, irrespective of the time of feeding, regardless of the accumulation of the β-subunit. The 7 S (mostly composed of β-conglycinin) and 11 S globulins (mostly glycinin, which was another major component of storage protein) contained N from 5 mM 15NO3 - at a similar level, about 60% of total N. These results indicate that a) the temporary supply of a high level of NO3 - effectively promoted β-subunit accumulation in N-deficient plants when feeding was carried out after 28 DAF, and b) incorporation of fed NO3 - was similar between 7 Sand 11 S globulins.

Seed storage protein composition of non-nodulating soybean ( Glycine max (L.) Merr.) and its influence on protein quality

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

Sulfur Availability, Cotyledon Nitrogen:Sulfur Ratio, and Relative Abundance of Seed Storage Proteins of Soybean

The nutritional value of soybean [Glycine max (L.) Merr.] seed protein could be enhanced by increasing its concentration of the S‐containing amino acids, methionine and cysteine. Two greenhouse pot studies and one field study were conducted with soybean grown under varying levels of S availability to observe the relationship between S availability, seed S content, and relative abundance of poor and high quality storage proteins. Abundances of the [β‐subunit of β‐conglycinin (poor quality) and of glycinin (high quality) seed storage proteins were determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Cotyledon‐S concentration more than doubled, and the N:S ratio of the seed decreased sharply (from about 40‐20 g N g S−1), as S availability increased from 12 to 62 mg available S per plant in the first greenhouse trial. The amount of the poor‐quality [β‐subunit of β‐conglycinin was linearly related to the N:S ratio of cotyledon tissue and varied from less than 15 up to 40% of storage proteins. On the other hand, the high‐quality glycinin fraction of storage protein showed a linear, negative relation to N:S ratio of cotyledon tissue and decreased from 60 to less than 30% of storage proteins as the N:S ratio increased under S stress. Even in high S environments the β‐subunit of β‐conglycinin comprised 10% or more of total storage proteins. Since poor quality storage protein was synthesized even in high S environments, we hypothesize that the plant's ability to reduce sulfate and synthesize S‐containing amino acids during seed filling may be a factor limiting soybean protein quality.

Regulation of accumulation of β-subunit of β-conglycinin in soybean seeds by nitrogen

Extract The storage protein of soybean [Glycine max (L.)] seed mainly consists of glycinin, composed of acidic (38 and 45 kDa) and basic (22 kDa) subunits (Kitamura et al. 1976), and β-conglycinin composed of α′- (75 kDa), α- (72 kDa), and β-(52 kDa) subunits (Thanh and Shibasaki 1978).

Inheritance of the .ALPHA.-subunit Deficiency of .BETA.-conglycinin in Soybean(Glycine max L. MERRILL) Line Induced by .GAMMA.-ray Irradiation.

We successfully induced a mutant soybean having a very low level of β-conglycinin. This mutant was characterized by the lack of the α - and α-subunits and by the low level of the β-subunit of β-conglycinin. We named this original mutant line 'T Iine'. In this paper, we report the mode of inheritance of the absence of the α-subunit and the genetic relationships among genes for the α-subunit and other subunits, including the α-subunit of β-conglycinin and the A4-subunit of glycinin. Eight crosses were made among the mutant 'T Iine' and other soybean lines producing the α-subunit. All the F1 seeds obtained from these crosses produced theα-subunit. The segregations for the presence or absence of the α-subunit were observed in the F2 seeds derived from the F1' Electrophoretic analyses of the F1 and F2 seeds revealed that the absence of the α-subunit was recessive to the presence and that the presence or absence of the α-subunit was controlled by a single allele.

Lack of β-subunit of β-conglycinin in non-nodulating isolines of soybean

Abstract Soybean seed storage protein is a very important protein source for human and livestock, and its amino acid composition is well-balanced except for the low level of sulfur amino acids, methionine, and cysteine. β-Conglycinin (7S globulin) and glycinin (11S globulin) are the major storage proteins in soybean seeds, and β-conglycinin consists mainly of α′, α, and β-subunits and glycinin consists of acidic and basic subunits. It is generally recognized that the sulfur amino acid content is higher in the following sequence: acidic subunit >; basic subunit> α′-subunit >; α-subunit >; β-subunit (Wilson 1987).

Isolation and characterization of a genomic clone encoding the ?-subunit of ?-conglycinin

β-Conglycinin, and abundant storage protein in soybean (Glycine max (L.) Merr.) seeds, is a trimeric protein consisting of various isomers containing the three subunits, α', α and β. Accumulation of the β-subunit is unique because it appears to be regulated by a variety of developmental and environmental signals. In this paper we describe the isolation and characterization of a genomic clone encoding the β-subunit of β-conglycinin. The genomic clone was characterized by restriction-enzyme mapping and partial DNA sequence analysis, by immunoprecipitation of a hybrid-selected invitro translation product, and by RNA blot hybridization reactions. An mRNA of approx. 1700 nucleotides hybridized to an internal 2-kilobase (kb) region of this 4.4-kb cloned DNA restriction fragment and was translated to yield a polypeptide with an approximate molecular weight of 48 kilodalton. This polypeptide is immunoprecipitable by antibody against β-conglycinin and is of appropriate size to represent the precursor polypeptide of the β-subunit. When this sequence was used as a probe in RNA blot hybridization experiments, the β-gene transcript was first detected by stage K and accumulated through stage O during soybean seed development, coincident with appearance of the β-subunit. Partial DNA sequence analysis of the 5' end of the gene confirmed that the isolated gene encoded a β-subunit, based upon the previously reported amino terminal sequence for this protein. Genomic DNA blot hybridization analyses indicate that multiple DNA restriction fragments are highly homologous to this cloned β-gene sequence.

Expression of the ?-subunit of ?-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein β-conglycinin, encoded by a multigene family. β-Conglycinin consists of three subunits; α', α, and β. A genomic clone for a β-subunit of β-conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this β-subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The β-subunit expressed in seeds of petunia and tobacco was recognized by anti-β-conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the β-subunit were produced. There was approximately a twofold variation in the accumulation of the β-subunit protein in the mature seeds of transgenic petunia plants, each containing a single β-subunit gene. However, the level of protein accumulation in mature seeds and the amount of β-subunit mRNA in developing seeds was not correlated. Accumulation of the β-subunit protein in transgenic seeds was less than the α'-subunit protein that accumulated in transgenic petunia seeds containing a single α'-subunit gene and less than the amount of the β-subunit in mature soybean seeds which contain 8-13 β-subunit genes. In transgenic tobacco plants, the accumulation of the β-subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.

Detection and characterization of a new β-conglycinin from soybean seeds

A new protein has been isolated from the reserve proteins of the seeds of soybean ( Glycine max) which is particularly deficient in methionine and cysteine. The protein dissociated in sodium dodecyl sulfate into a single polypeptide, M r 48,000. The amino acid composition, N-terminal leucine and mobility on gel electrophoresis of this polypeptide all were indistinguishable from the β-subunit of β-conglycinin. In its nondissociated form, the protein behaved as a trimer of M r, 137,000 ± 4000. Its sedimentation coefficient at ionic strength 0.5 was 7.5 S and it possessed antigenic determinants in common with β-conglycinin. This protein therefore has the properties of a new isomer of β-conglycinin—a homogeneous trimer of β subunits.