Structural homology among the major 7s globulin subunits of soybean seed storage proteins

The soybean seed 7S globulin subunits, i.e. α, α′, β and γ-subunits of β-conglycinin, the γ-conglycinin subunit and the HI/HII and LII subunits of basic 7S globulin were purified and the NH₂-terminal amino acid sequences of all these subunits except the γ-subunit of β-conglycinin were determined. Only the NH₂-terminal regions of the α and α′-subunits showed high sequence homology. However, sequencing of tryptic peptides from the seven subunits revealed that internal region sequences were highly homologous among the four subunits of β-conglycinin. In contrast to the β-conglycinin subunits, no sequence homology was found among the other subunits. On the basis of these results, the major 7S globulin fraction is considered more heterogeneous in primary structure than another major globulin fraction, 11S globulin (glycinin), in soybean seeds.     

Characterization of the 12S globulin complex of Brassica napus. Evolutionary relationship to other 11-12S storage globulins

Cruciferin (12S globulin) is the major seed protein in Brassica napus (oil seed rape). It is synthesized during seed development and consists of six subunit pairs. Each of these pairs is synthesized as a precursor containing one alpha and one beta chain. At least three different precursors exist (P1-3), giving rise to four different mature subunits (cru1-4). Several cruciferin clones were isolated from a seed mRNA cDNA library. Comparison of the deduced amino acid sequences of these clones to amino acid sequences of purified cruciferin chains and peptides identified them as coding for cru2/3 and cru4 subunits. From the amino acid sequences deduced from two overlapping cDNA clones, the precursor of the cru4 subunit was shown to consist of 465 amino acid residues. Comparison of cruciferin and cruciferin-related sequences from B. napus and Arabidopsis thaliana, respectively, suggested that early during evolution the Brassicaceae family only possessed two types of 11-12S globulin genes, like the present-day Fabaceae.     

Isolation of legumin-like protein from Phaseolus aureus and Phaseolus vulgaris

An 11S seed globulin has been isolated from Phaseolus aureus and P. vulgaris by zonal isoelectric precipitation and the MWs of the constituent subunits determined. The protein of P. vulgaris occurs in the protein body fraction and its chemical composition, including the N-terminal amino acids and amino acid composition has been determined. The similarity between the 11S globulin of the two Phaseolus spp. and legumin from other leguines is discussed.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

The major albumin proteins from pea (Pisum sativum L). Purification and some properties.

A scheme is described for the fractionation of pea (Pisum sativum) albumin proteins. By using this scheme, two closely related major albumin proteins have been isolated and purified to homogeneity. The larger protein, designated PMA-L, has Mr approximately 53 000 and consists of two 25 000-Mr subunits, whereas the smaller, PMA-S, has Mr approximately 48 000 and contains two 24 000-Mr subunits. There was no evidence of mixed dimers of the two subunit sizes, despite their close homology as judged by immunological crossreaction, amino acid composition, N-terminal amino acids, tryptic-peptide mapping and CNBr-cleavage products. Both proteins contained significant amounts of sulphur amino acids. The proteins were shown to be located in the soluble cytosol fraction of cotyledon cells and are not significantly degraded on seed germination. Preliminary screening indicates the presence of homologous major albumin proteins in at least three different, though closely related, legume species.     

Protein subunits and amino acid composition of wild lentil

Three wild species of lentil, Lens orientalis, L. ervoides and L. nigricans were investigated for protein subunits of the albumin protein fraction (APF), globulin protein fraction (GPF) and for protein and free amino acid composition. The APF and GPF formed 12.7–16.8 % and 34.7–49.0 %, respectively, of the meal nitrogen. SDS-PAGE showed APF to contain 15 to 20 major and a similar number of minor protein subunits ranging in M_r at least from 14 400 to 94 000. The GPF was also heterogenous and contained some subunits having M_r similar to APF subunits but none < 15 000. The three wild lentil species were distinguishable by their protein subunit composition. The protein amino acid composition of the wild species was identical and similar to that of the cultivated lentil. The wild species, like the cultivated species (L. culinaris), contained major amounts of free arginine, glutamic and aspartic acids, serine and a number of unidentified amino acids. L. orientalis, L. nigricans and the cultivated lentil contained two acidic and two basic unidentified amino acids. However, L. ervoides was distinctly different in that it contained only the two acidic plus one neutral unidentified amino acid, but none of the two basic unidentified amino acids.     

Changes in salt-soluble proteins of rice during grain development

Salt-soluble proteins, albumin and globulin, were prepared from dehulled rice (Oryza sativa L., line IR1541-76-3) during grain development. Albumin and globulin progressively increased during grain development up to about 12 days after flowering (DAF) and then decreased slightly during grain desiccation. Free amino N was maximum at 10 DAF. Total protein and glutelin-prolamin (by difference) continued to increase up to 20 DAF. Aminogram of total protein and globulin showed a progressive decrease in lysine and threonine among the essential amino acids. Albumin showed a similar trend except for the lesser change in lysine content. Disc gel electrophoresis showed a maximum of four major and six minor protein bands for albumin and only one major and three minor bands for globulin. Sodium dodecyl sulfate-gel electrophoresis revealed three major polypeptide subunits for albumin with MW of 11 000, 8 500 and 16 000, and two for globulin with MW of 20 000 and 12 000.     

Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds

Key message

A platform of gene silencing by amiRNA had been established in fertile transgenic soybean. We demonstrated that knockdown of storage protein shifted the distribution of nitrogen sources in soybean seeds.

Abstract

Artificial microRNAs (amiRNAs) were designed using the precursor sequence of the endogenous soybean (Glycine max L. Merrill) miRNA gma-miR159a and expressed in transgenic soybean plants to suppress the biosynthesis of 7S globulin, which is one of the major storage proteins. Seed-specific expression of these amiRNAs (amiR-7S) resulted in a strong suppression of 7S globulin subunit genes and decreased accumulation of the 7S globulin subunits in seeds. Thus, the results demonstrate that a platform for gene silencing by amiRNA was first developed in fertile transgenic soybean plants. There was no difference in nitrogen, carbon, and lipid contents between amiR-7S and control seeds. Four protein fractions were collected from defatted mature seeds on the basis of solubility at different pH to examine the distribution of nitrogen sources and compensatory effects. In the whey and lipophilic fractions, nitrogen content was similar in amiR-7S and control seeds. Nitrogen content was significantly decreased in the major soluble protein fraction and increased in the residual fraction (okara) of the amiR-7S seeds. Amino acid analysis revealed that increased nitrogen compounds in okara were proteins or peptides rather than free amino acids. Our study indicates that the decrease in 7S globulin subunits shifts the distribution of nitrogen sources to okara in transgenic soybean seeds.     

Combined 2D electrophoretic approaches for the study of white lupin mature seed storage proteome

Seed proteome analysis by 2D IEF/SDS-PAGE techniques is challenging for the intrinsic difficulties related to quantitative disparity of the seed proteins, i.e. storage and non-storage proteins, their polymorphic nature, the extensive post-translational modifications and the paucity of deposited primary structures available. Conversely, 2D maps of seed proteomes can be extremely useful for a number of fundamental and applied investigations. In this work, we have used a combination of two experimental approaches to identify the main protein components of an emerging protein-rich legume seed, that is white lupin seed (Lupinus albus, L.). One is the canonical proteomic approach including 2D electrophoretic separation and mass spectrometry of selected trypsin-digested polypeptides; the other approach is a group comparative 2D electrophoretic analysis of cotyledonary protein families. To this second purpose, the three main families of lupin seed proteins, namely alpha-conglutins, the 11S globulin fraction, beta-conglutins, the 7S globulin fraction, and gamma-conglutin, a basic 7S protein, were isolated by conventional biochemical techniques and their 2D reference maps were compared with the total protein map. With the first approach 37 out of 40 spots, making up about 35% of total spot volumes in the 2D map, were found to belong to the main seed protein families. Thanks to cDNA-deduced lupin storage protein sequences, determined on purpose and deposited, most of the identification statistical parameters were very good. Moreover, it was possible to identify several endogenously proteolysed subunits in the map. The second comparative approach, beside confirming these attributions, allowed to allocate 124 polypeptides within the three main lupin protein families. These two approaches proved to be mutually validating and their combined use was effective for the establishment of a seed proteome map even in the case of sequence and protein post-translational processing lack of information. The results obtained also extend our knowledge of the seed storage protein polymorphism of white lupin.     

Effects of exogenous methionine on storage protein composition of soybean cotyledons cultured in vitro

Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental l-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S beta-subunit gene. Concomitant with the absence of the beta-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of beta-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

Storage Protein Composition of Soybean Cotyledons Grown In Vitro in Media of Various Sulfate Concentrations in the Presence and Absence of Exogenous l-Methionine

Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental l-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S β-subunit gene. Concomitant with the absence of the β-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of β-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.     

Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency

Soybean ( Glycine max [L.] Merr.) seeds are rich in protein, most of which is contributed by the major storage proteins glycinin (11S globulin) and beta-conglycinin (7S globulin). Null mutations for each of the subunits of these storage proteins were integrated by crossbreeding to yield a soybean line that lacks both glycinin and beta-conglycinin components. In spite of the absence of these two major storage proteins, the mutant line grew and reproduced normally, and the nitrogen content of its dry seed was similar to that for wild-type cultivars. However, protein bodies appeared underdeveloped in the cotyledons of the integrated mutant line. Furthermore, whereas free amino acids contribute only 0.3-0.8% of the seed nitrogen content of wild-type varieties, they constituted 4.5-8.2% of the seed nitrogen content in the integrated mutant line, with arginine (Arg) being especially enriched in the mutant seeds. Seeds of the integrated mutant line thus appeared to compensate for the reduced nitrogen content in the form of glycinin and beta-conglycinin by accumulating free amino acids as well as by increasing the expression of certain other seed proteins. These results indicate that soybean seeds are able to store nitrogen mostly in the form of either proteins or free amino acids.     

Proteolysis of the main storage protein of buckwheat seeds at the early stage of germination

The changes in the main storage protein of seeds of buckwheat ( Fagopyrum esculentum Moench cv. Shatilovskaya 5), 13S globulin, were studied during seed germination. During the first three days of germination the 13S globulin is subjected to a limited proteolysis, which consists in the splitting of some of its subunits into large polypeptide fragments. Insignificant changes in the sedimentation coefficient of the 13S globulin during the first days of germination as well as immunochemical data, indicate that the limited proteolysis of the 13S globulin does not cause any major changes in its structure.
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination.     

Homology among 3S and 7S Globulins from Cereals and Pea

The globulins from wheat caryopses were found to consist primarily of protein sedimenting at approximately 3S and 7S. These proteins displayed a molecular weight distribution similar to that of the purified vicilin-like fractions from oat and pea, with variations occurring in the isoelectric points and relative quantities of their major subunits. concanavalin A Sepharose chromatography suggested that the major polypeptides of the wheat (3S + 7S) fraction are glycosylated. Western blot analysis using antioat (3S + 7S) globulin immunoglobulin G revealed the vicilins from pea and the globulin fractions of oat, wheat, barley, rye, corn, and rice to contain immunologically homologous polypeptides. Major groups of polypeptides were shared by all the cereals and pea, including subunits of approximately 75, 50, 40 kilodaltons and 20 to 25 kilodaltons. These results indicate that legume-like 3S and 7S globulins have been conserved and are being expressed in cereals.     

Characterization of the Major Protein Component from Aleurone Cells of Barley (Hordeum vulgare L.)

Comparison of the SDS-PAGE patterns of salt-soluble proteinsfrom aleurone protoplasts, enriched aleurone layers preparedby pearling, and isolated embryos of mature barley showed threegroups of bands that reacted with antiserum raised against the7S globulins of oat embryos. These had M_rs of about 50 000,40 000 and 25 000. The enriched aleurones also contained a thirdgroup of immunoreactive bands (M_r about 70 000), which did notco-purify when the proteins were fractionated by ammonium sulphateprecipitation, ion exchange chromatography and immunoaffinitychromatography. The purified protein gave a single sharp peakon RP-HPLC, and contained a fourth minor group of subunits ofM_r about 20 000, in addition to those of M_rs about 50 000, 40000 and 25 000. The holoprotein and the ‘major groups’ of subunitsall had similar major N-terminal amino acid sequences whichwere related to the N-terminal amino acid sequences of pea andbean vicilins, and to sequences in the putative N-terminal regionsof the mature 7S ¹-globulins of cottonseed, confirming the homologywith these groups of proteins. Key words: Barley, seeds, 7S globulin, vicilin, ¹-globulin, amino acid sequence, protein homology     

Characterization of a cDNA clone encoding a Brassica napus 12 S protein (cruciferin) subunit. Relationship between precursors and mature chains

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.     

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.     

抗、感绿豆象绿豆种子差异蛋白的分离与鉴定

为寻找抗绿豆象绿豆种子中的重要抗虫成分,以抗虫绿豆晋绿7号、B20及感虫绿豆潍绿2117为试验材料,采用蛋白质双向电泳(2-DE)和质谱技术,对抗、感虫绿豆中差异蛋白及功能进行了鉴定与分析。结果表明,抗、感虫绿豆中差异蛋白表达量超过2.5倍的点共有15个。其中6个蛋白点通过数据库得到了成功鉴定,涉及3种蛋白质,分别为8S球蛋白(α亚型和β亚型)、核酮糖1,5-二磷酸羧化酶/加氧酶(RuBis CO)亚基结合蛋白和合成淀粉酶与胰蛋白酶两种抑制剂的前体多肽链。蛋白点B49(即8S球蛋白α亚型)和B31(RuBis CO亚基结合蛋白)在抗、感虫绿豆中的差异表达量分别达到10 000倍和23倍。抗虫绿豆中8S球蛋白α亚型及β亚型、RuBis CO伴侣蛋白及胰蛋白酶抑制剂的前体作为抗虫物质影响了绿豆象的生长发育甚至导致其死亡,与抗虫性的量效关系及联合效应还需进一步验证。     

Identification of Homologous Globulins from Embryos of Wheat, Barley, Rye and Oats

Total globulins from embryos and endosperms of barley, wheat,rye, and oats were separated by SDS-PAGE under reducing andnon-reducing conditions. The preparations from embryos of allfour cereals contained major groups of bands with M_r's of 50-60000,which were not affected by reduction. These have been characterizedpreviously from oats and shown to correspond to subunits ofthe 7S storage globulin. Immunochemical relationships betweenthese bands (and others with M_r's between 40000 and 70000) weredemonstrated by immunodiffusion and ‘Western Blotting’using antiserum raised against the major subunits of the oat7S globulins. The 7S globulins were also prepared from hand-dissectedembryos of the four cereals using sucrose density ultracentrifugation.Their amino acid compositions were broadly similar, but differedfrom those of the 7S vicilins of legumes. It is concluded thatstructurally-related 7S globulins are present in the embryosof the four species of cereals. Key words: Homologous globulins, embryo, wheat, barley, rye, oats