Soybean Seed Storage Protein Research Articles

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean ( Glycine max L.) storage proteins are composed mainly of two major components, β-conglycinin and glycinin. Electrophoretic variants of the β subunit of β-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as β* and A3*, respectively. β* and A3* exhibited higher and lower mobilities, respectively, than the common β subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of β* and A3* were completely identical to the previously reported sequences of the β subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of β* or A3*. Furthermore, five clones of β* or β and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common β subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of β* and A3*.

Regulatory Mechanism of Soybean Seed Storage Protein Accumulation by Nitrogen Nutrition (The Progress Awards)

Regulatory Mechanism of Soybean Seed Storage Protein Accumulation by Nitrogen Nutrition (The Progress Awards)

Protein sorting and expression of a unique soybean cotyledon protein, GmSBP, destined for the protein storage vacuole.

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and beta-conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, beta-conglycinin (alpha/alpha' subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.

Differential tissue-specific response to sulfate and methionine of a soybean seed storage protein promoter region in transgenic Arabidopsis.

Expression of the gene encoding the beta subunit of beta-conglycinin, a major soybean seed storage protein, is upregulated by sulfur deficiency and downregulated by methionine (Met). The tissue-specificity of these regulatory mechanisms was studied using a sulfate-responsive region (beta(SR)) from the beta subunit gene promoter. Transgenic Arabidopsis thaliana lines were generated carrying a green fluorescent protein (GFP) reporter gene under control of the cauliflower mosaic virus 35S RNA promoter with a tandem repeat of the beta(SR) element, referred to as the P35S::beta(SR)x3: GFP transgene. Upregulation of P35S::beta(SR)x3:GFP by sulfur deficiency was strongest in leaf margins, where symptoms of sulfur deficiency first appear. P35S::beta(SR)x3:GFP was also upregulated at 2 d after a medium shift from sulfur-sufficient to sulfur-deficient conditions, suggesting that the chimeric promoter is an efficient indicator of sulfur nutritional status. Analysis of transgene expression in a Met-overaccumulating mto1-1 mutation background revealed that the beta(SR) region carries sufficient information for downregulation of promoter activity by Met in developing seeds, but not in young rosettes. Comparisons with another transgenic line, in which the full-length beta promoter is active in non-seed tissues, also suggested that at least two separate tissue-specific mechanisms exist for the downregulation of the beta promoter by Met.

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.

Structure and characterization of the gene encoding alpha subunit of soybean beta-conglycinin.

beta-conglycinin, a soybean seed storage protein, is comprised of three different subunits, a, alpha', and beta. Several candidates for the alpha subunit gene have been isolated, however, the structure of the alpha subunit gene has not been completely determined. Accordingly, it was also unknown which of the gene candidates are functionally active. Here, we have determined the nucleotide sequence and transcription start site of the alpha subunit gene, and compared the structural components with those of the other subunits or other seed protein genes. The a subunit gene, which is located on a 7.6-kb EcoRI fragment, was composed of six exons that had the same organization as those for the alpha' subunit gene. Within a 400 bp upstream region of the transcription start site, four regions (designated as boxes I, II, III, and IV) were found to be conserved among the alpha, alpha', and other seed protein genes. Genomic Southern blot analysis of soybean varieties lacking the alpha subunit gene candidate indicated that the gene characterized in this paper actually encodes the a subunit and is functionally active. In addition, these experiments revealed the presence of an additional gene which is also responsible for the expression of the a subunit.

Biochemistry and Molecular Biology of Soybean Seed Storage Proteins

Soybean (Glycine max[L.] Merr.) is one of the principle field crops grown in the United States. From a crop with no significant economic value at the turn of this century, soybeans have made remarkable strides in U.S. agriculture, claiming the stature of the number two U.S. cash crop. Soybean is an important source of edible vegetable oil and protein throughout the world and is used in a multitude of food and industrial applications. Even though soybeans are a rich source of protein for livestock and humans, the nutritional quality of soybean proteins is not optimal. Some of the problems associated with soybean proteins include (1) presence of anti-nutritional factors such as trypsin inhibitor, (2) undesirable beany flavor, (3) elicitation of allergic reactions in susceptible individuals, (4) poor digestibility of soybean proteins, and (5) deficiency in sulfur-containing amino acids. As a consequence, concerted efforts are underway to improve the overall nutritive value of soybean proteins by both classical plant breeding and molecular biological approaches. This review article summarizes the current knowledge on the biochemistry and molecular biology of soybean seed storage proteins. Recent advances in the genetic improvement of amino acid composition of seed storage proteins are highlighted. The review also includes some recent achievements in modifying soybean seed composition.

Differential Accumulation of Soybean Seed Storage Protein Subunits in Response to Sulfur and Nitrogen Nutritional Sources

Soybean (Glycine max [L.] Merr.) seed storage proteins consist of subunits that differ in amino acid profile, the β-subunit of 7S protein being essentially devoid of the S-containing amino acids, methionine and cysteine. Our objective was to examine the interaction of N and S nutrition on the relative abundance of these storage protein subunits in soybean seed. ‘Kenwood’ soybean was grown in hydroponic culture, and during vegetative growth (V2–R4.5) N was provided as 5 mM KNO3 to plants grown under sulfur-deficient (0.004 raM Na2SO4) or sulfur-sufficient (0.4 mM Na2S04) conditions. During seed fill (R4.5–R7) N was supplied as 5 mM KNO3 or 2.5 mM urea. Each N group was given S treatments of 1) no sulfur, 2) 0.4mM Na2SO4, 3) 0.2 mM L-cystine, or 4) 0.4 mM L-methionine. Effects on seed protein quality of S deficiency during vegetative growth were essentially overcome by supplying sulfate as late as R4.5. Total protein and seed storage protein were increased with urea as N source, but urea also increased the β-subunit. Provision of reduced S as methionine essentially suppressed β-subunit production, but cystine did not, suggesting that cystine did not influence methionine level in the seed. We also report the accumulation of two as yet unreported proteins which occur at extremes of S nutrition : (1) a putative β-subunit of 7S protein occurring in the embryonic axis under S-deficiency ; and (2) a ca. 14kD protein in cotyledon tissue under provision of L-methionine. Though S and N did interact to a limited extent to influence seed protein composition, major effects were from S or N acting individually.

Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin, a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a β-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the β-sheet (6%) and random coil (12%) to the α-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the film-forming process leading to the formation of intermolecular hydrogen-bonded β-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded β-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the film state, intermolecular hydrogen bonding between segments of β-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films.

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.

Analysis of the Mechanisms Underlying Regulation of the Soybean Seed Storage Protein Gene Expression in Response to Sulfur Nutritional Conditions

Analysis of the Mechanisms Underlying Regulation of the Soybean Seed Storage Protein Gene Expression in Response to Sulfur Nutritional Conditions

Nutritional Control of Soybean Seed Storage Protein

There is interest in increasing the protein concentration of soybean [Glycine max (L.) Merr.] seed. Potentially, this could change its protein composition and perhaps its value as a livestock feed. Our objective was to examine the change in protein composition of soybean seed with change in protein concentration as influenced by nitrogen source supplied. ‘Harper’ soybean was grown in hydroponic culture and supplied with different nitrogen sources (KNO3, NH4NO3N2, urea) during pod filling. Protein quality was monitored as a function of soybean seed storage subunit composition. With N2 fixation as sole N source, the concentration of total seed protein decreased about 6%, storage protein 5%. Each seed storage protein subunit decreased, but the concentration of [β‐conglycinin, especially the sulfur‐poor [β‐subunit, decreased more than glycinin. Seed storage protein concentration was increased up to 4% by substituting reduced‐N for NO−3. The increased protein concentration resulted from a disproportionate increase of the β‐subunit of β‐conglycinin. The relative abundance of α‐ and α′‐subunits was not changed by N form. Thus, N form seems to control, at least in part, the relative portion of the β‐subunit of [β‐conglycinin, and consequently affects the 11S/7S ratio of seed storage protein. This change in composition with increased concentration diminishes the protein quality because the [β‐submit lacks sulfur‐amino acids. It is noteworthy that the concentration of glycinin remained constant while the total seed protein concentration increased. We think these variations in seed storage protein composition result from the relative abundance of S‐ and N‐metabolites available to developing soybean seed.

Characterization of 30-kDa Fragments Derived from β-Conglycinin Degradation Process during Germination and Seedling Growth of Soybean

The degradation process of beta-conglycinin, a vicilin-type glycosylated storage protein of soybean seeds, during germination and seedling growth was examined by concanavalin A blotting combined with polyacrylamide gel electrophoresis. We detected and analyzed the structures of key intermediary fragments of 30 kDa derived from beta-conglycinin degradation, they were proved to be single-domain type subunits of beta-conglycinin. We show here a degradation process of beta-conglycinin: beta-conglycinin is subjected to limited proteolysis at exposed regions on the molecular surface, like domain junctions, generating 30-kDa single-domain fragments before non-specific proteolysis.

Effects of exogenous ABA application on sulfate and OAS concentrations, and on composition of seed storage proteins in in vitro cultured soybean immature cotyledons

Composition of seed storage proteins in in vitro cultured cotyledons is known to be affected by exogenous application of abscisic acid (ABA) and sulfur deficiency. In this paper, we analyzed effects of exogenous ABA on regulation of soybean seed storage protein accumulation in relation to that of sulfur deficiency. When exogenous ABA was applied at 10 μM and 100 μM, accumulation of the β subunit of β-conglycinin, a major seed storage protein, increased, whereas that of glycinin, another major seed storage protein, decreased. Free sulfate concentrations in cotyledons were not significantly affected by application of 10 μM ABA. but doubled by application of 100 μM ABA. As we have previously shown that concentrations of O-acetyl-L-serine (OAS), a precursor of cysteine biosynthesis, in in vitro cultured soybean cotyledons increased under sulfur deficiency, effects of exogenous ABA application on OAS concentration in soybean cotyledons were determined. The concentrations of OAS increased in accordance with the increase of exogenous ABA concentrations. These results suggested that exogenous application of ABA induces changes in soybean storage protein composition through the increase of OAS concentration.

Effects of Sulfate Concentrations on the Expression of a Soybean Seed Storage Protein Gene and its Reversibility in Transgenic <italic>Arabidopsis thaliana</italic>

Transgenic expression of genes encoding the alpha' and beta subunits of beta-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.), was analyzed in Arabidopsis thaliana (L.) Heynh. under conditions of sulfate deficiency. Temporal patterns of expression of both the intact beta subunit gene and the beta subunit gene promoter fused to the beta-glucuronidase (GUS) gene are similar in soil-less cultures using rockwool, suggesting that the response to sulfate deficiency is regulated mainly at the level of transcription. In hydroponic cultures with various concentrations of sulfate, expression of both the intact beta subunit gene and the beta subunit gene promoter-GUS fusion gene were negatively correlated to increased sulfate concentrations in the culture medium. Transfer of transgenic A. thaliana plants carrying the beta subunit gene promoter-GUS fusion from sulfate-deficient to sulfate-sufficient control medium caused GUS activity in developing siliques to be repressed within two days. A reverse shift, where the plants were transferred from the control to sulfate-deficient medium, caused GUS activity to become higher than that in seeds of the control plants within two days. These results indicate that the expression of the beta subunit gene promoter responds rapidly to changes of sulfate availability.

Expression of Soybean Seed Storage Protein Genes in Transgenic Plants and Their Response to Sulfur Nutritional Conditions

Summary β -Conglycinin, the 7S seed storage protein of soybean ( Glycine max [L.] Merr.), is comprised mainly of three subunits, designated α ' , ex and β . Among these subunits, expression of the β subunit is unique because its accumulation is enhanced under sulfate deficiency in vivo and repressed by exogenously applied L-methionine in cotyledon cultures of soybean. Transgenic Arabidopsis thaliana (L.) Heynh. strains carrying either intact gene encoding the β subunit, or β -glucuronidase fusion were constructed and the effects of sulfur nutritional conditions were analyzed. Expression of the transgene was enhanced under sulfate deficiency and repressed when the siliques of transgenic A. thaliana plants were cultured in the presence of L-methionine. The results indicate that response to sulfur nutritional conditions are conserved in transgenic A. thaliana . By using an A. thaliana mutant, designated mto1-1 , that overaccumulates free methionine, activity of the β subunit gene promoter was shown to be repressed in the seeds. These transgenic and mutant A. thaliana plants will be useful in analyzing the plants' response to nutritional conditions.

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).

Crystallization and preliminary X-ray analysis of soybean proglycinins modified by protein engineering.

Glycinin is one of the most abundant storage proteins in soybean seeds. We earlier reported the preparation of proglycinins modified by protein engineering to improve food functions. Crystals of the modified proglycinins (delta I, delta V8, IV + 4Met, V + 4Met, Gly12, and Ser88) expressed in Escherichia coli were grown, each under different suitable crystallization conditions. The crystals of delta I, V + 4Met, Gly12, and Ser88 diffracted X-rays sufficiently for crystallographic analysis. delta I, Gly12, and Ser88 crystals were tetragonal, space group P4(1) or P4(3), and with unit cell dimensions a = b = 114.3-115.9 A and c = 145.1-146.1 A. V + 4Met crystals were monoclinic, space group P2, and with unit cell dimensions a = 118.7 A, b = 78.1 A, c = 109.9 A, and beta = 119 degrees. The number of promoters per asymmetric unit of all of the crystals of these four modified proglycinins was about 3. This value is consistent with proglycinins being trimers. These data indicated that most of the modified proglycinin crystals examined here could be studied by X-ray crystallography to elucidate the relationships between the structure and the functional properties of glycinin at molecular level.

Differential Regulation of Soybean Seed Storage Protein Gene Promoter-GUS Fusions by Exogenously Applied Methionine in Transgenic <italic>Arabidopsis thaliana</italic>

Differential Regulation of Soybean Seed Storage Protein Gene Promoter-GUS Fusions by Exogenously Applied Methionine in Transgenic <italic>Arabidopsis thaliana</italic>

Tissue-specific and temporal regulation of a ?-conglycinin gene: roles of the RY repeat and other cis-acting elements

Upstream regulatory sequences (URS) of the gene that encodes the alpha' subunit of beta-conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623-631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the alpha' subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the alpha' promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the alpha' promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the alpha' URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.