Antigenic relationship of legume seed proteins to the 7S seed storage protein of soybean

Extracts enriched for globulin proteins were prepared from the seeds of a large number of legume species and were tested for homology to antisera prepared against the glycosylated 7S seed storage protein of the soybean (Glycine max). Electrophoretic identification and subsequent analysis of proteins precipitated with 7S antisera was useful at relatively short taxonomic distances, particularly within the tribe Phaseoleae, to which G. max belongs. Glycine and most other members of the subtribe Glycininae are unusual within the Phaseoleae in having high molecular weight ($\text{> 70 000}$ dalton) subunit polypeptides. Seeds from other plants representing other subtribes of the Phaseoleae also contained proteins that cross-reacted with the G. max antisera; the molecular weights of these proteins varied from 30 000 to nearly 90 000 daltons. Homology was detected across a wider range of legume tribes within the subfamily Papilionoideae by enzyme-linked immunosorbent assay (ELISA). The results of these experiments suggest both that the 7S proteins of these tribes are evolutionarily related and that at least some features of these apparently rapidly-evolving proteins are under relatively strong selectional constraint.     

Immunological relationships among the major seed proteins of cereals

The structural relationships among the major seed proteins of cereals was evaluated by Western blot analyses using antibodies raised against the wheat gliadin, rice glutelin acidic and basic subunits, and rice prolamine polypeptide. Consitent with the conservation of the primary sequences of these proteins, antibodies to the acidic and basic glutelin subunits cross-reacted with homologous polypeptides from oat as well as pea. The rice glutelin antibodies did not react with the major seed proteins from barley, rye, maize and sorghum. Antibodies raised against the acidic glutelin subunit reacted with the wheat glutenins but antibodies to the basic glutelin subunit did not. A comparison of the published primary sequences of a high molecular weight glutenin and rice glutelin showed little similarity except for a conserved peptide with the motif arg-gln-leu-gln-cys. The possible significance of this conserved element shared by these widely different proteins is discussed. Similar studies with the wheat gliadin antibody showed immunologically related components in plants of the subfamily Festucoideae except for rice. Antibodies raised against the rice prolamine recognized only the rice prolamine, indicating that this polypeptide was structurally distinct from other cereal prolamines. Overall, these results support and help clarify the evolutionary relationship of the cereals.     

种子盐溶球蛋白的结构特征

种子球蛋白一直是人类食物的主要来源,随着对种子球蛋白药理作用认识的不断深入,近年来研究重点已从单纯的序列转向结构的研究,种子球蛋白二级结构（如α－螺旋和β－折叠）存在较大程度的相似性,具有较大的保守性且与蛋白质的特殊功能关系密切,而三级结构之间差异明显,属易变异区,主要与蛋白质的一般功能相关。     

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, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta 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 beta* or A3*. Furthermore, five clones of beta* or beta 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 beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

The globulin seed storage proteins of flowering plants are derived from two ancestral genes

Summary The cDNA and/or genomic DNA sequences of 13 globulin storage proteins from flowering plants (angiosperms) are now known. They represent 8 genera, 5 families and 5 orders of plants and include one monocotyledonous species. Here, the coding nucleotide and amino acid sequences of these proteins are compared by dot matrix analysis and gross protein domains visualized by hydropathy analyses. The vestigial homologies visualized by these means indicate that all of the globulin storage proteins of flowering plants have emanated from 2 genes that existed at the beginning of angiosperm evolution.A curious polypeptide domain of 150–200 amino acids located near the N terminus is found in a globulin subgroup of 2 genera widely separated phylogenetically. The domain appears to have resulted from an ancient insertion that has been deleted in most of its descendant genes.     

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins

The wheat high molecular weight (HMW) glutenins are important seed storage proteins that determine bread-making quality in hexaploid wheat (Triticum aestivum). In this study, detailed comparative sequence analyses of large orthologous HMW glutenin genomic regions from eight grass species, representing a wide evolutionary history of grass genomes, reveal a number of lineage-specific sequence changes. These lineage-specific changes, which resulted in duplications, insertions, and deletions of genes, are the major forces disrupting gene colinearity among grass genomes. Our results indicate that the presence of the HMW glutenin gene in Triticeae genomes was caused by lineage-specific duplication of a globulin gene. This tandem duplication event is shared by Brachypodium and Triticeae genomes, but is absent in rice, maize, and sorghum, suggesting the duplication occurred after Brachypodium and Triticeae genomes diverged from the other grasses ~35 Ma ago. Aside from their physical location in tandem, the sequence similarity, expression pattern, and conserved cis-acting elements responsible for endosperm-specific expression further support the paralogous relationship between the HMW glutenin and globulin genes. While the duplicated copy in Brachypodium has apparently become nonfunctional, the duplicated copy in wheat has evolved to become the HMW glutenin gene by gaining a central prolamin repetitive domain.     

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.     

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.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

Characterization of 2S seed storage protein of Brassica campestris and its antigenic homology with seed proteins of other Cruciferae.

The low molecular weight seed storage protein of Brassica campestris has been isolated and its amino acid composition determined. Antibody raised against this low molecular weight protein has been used to compare the antigenic similarity between the low molecular weight storage proteins of different Cruciferae seeds by immunoprecipitation and Western blotting. These studies revealed the existence of antigenically homologous proteins of identical molecular weights in seeds of other Cruciferae but absent in some other dicots like mung bean and tobacco seeds.     

Characterization of olive seed storage proteins

At present little is known about olive seed storage proteins (SSPs). A better understanding of olive SSPs will be important for future biotechnology efforts. In the present study, we first developed a protocol relied on chloroform for preparing protein samples free of lipids from lipid-rich olive seeds. Then, we characterized olive SSPs by SDS-PAGE, N-terminal sequencing and immunoblot. Two smaller subunits (20 and 21.5 kD) of SSPs were purified to homogeneity and used for antibody production or N-terminal sequencing. N-terminal sequencing confirmed that major olive SSPs are 11S globulins. Moreover, the components and size distribution of SSPs are identical among several olive cultivars examined, suggesting that their synthesis is highly conserved in this species. Olive SSPs are soluble in aqueous alcohol, with limited solubility in water and dilute salt. Thus, despite their homology with globulins, olive SSPs are similar in solubility to prolamins and different from globulins in other dicot plants. Finally, the accumulation of olive SSPs during fruit maturation was examined. Our results revealed that the accumulation of SSPs is time-dependent and tissue-specific, and only 105 days after pollination (DAP), did individual components of SSPs synthesize substantially, and accumulate rapidly in large quantities over a short period of time. Our results suggest that a 36 kD protein is the precursor of olive SSPs, and 90–105 DAP seems to be a crucial transition period (from a precursor to mature subunits) for the accumulation of SSPs.     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

Structural homology between semidwarfism-related proteins and glutelin seed protein in rice (Oryza sativa L.)

Summary Two semidwarfism-related proteins, SRP-1 and SRP-2, were detected as major spots in a long-culm rice cultivar, Norin 29 and its semidwarf near-isogenic line, SC-TN1, respectively, by two-dimensional gel electrophoresis (2D-PAGE). The testcross showed that SRP-1 and SRP-2 are controlled by codominant alleles, Srp-1 and Srp-2, respectively, at a single locus Srp. This locus was considered to be closely linked with the semidwarfing gene locus sd-1. SRP-1 and SRP-2 were separated by 2D-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and sequenced by a gas-phase protein sequencer. The N-terminal amino acid sequences, however, could not be determined due to the blockage of the N-terminals of these proteins. After removal of the N-terminal residue with pyroglutamyl peptidase given to the membrane, the amino acid sequence in the N-terminal region was determined. The N-terminal and internal amino acid sequences of SRP-1 and SRP-2 were highly homologous with those of the glutelin -subunits of seed endosperm storage protein, which were deduced by the cDNA sequences. In the seed endosperms of Norin 29 and SC-TN1, a total of eight glutelin -subunits was identified by 2D-PAGE. The amino acid sequences in the N-terminal and internal regions of these proteins were determined. This experiment confirmed that SRP-1 and SRP-2 are almost identical in structure with the glutelin _5a- and _5b-subunits, respectively, which were identified in several organs such as endosperms, embryos, and leaves, unlike the other glutelin -subunits.     

Structural homology of storage proteins coded by the Hor-1 locus of barley (Hordeum vulgare L.)

Three C hordein fractions were prepared by ion-exchange chromatography of a total hordein preparation on carboxymethyl cellulose at pH 4.6 Polyacrylamide gel electrophoresis at pH 3.2 and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at pH 8.9 showed that each fraction contained a single major band. The apparent molecular weights of these were determined by SDS-PAGE as 58, 57, and 54,000. When compared by isoelectric focusing, however, the 58 and 57,000 components each separated into two major bands and the 54,000 component into four. Amino acid analysis showed that although the three fractions had similar compositions with high glutamate+glutamine (38–39%), proline (30–32%) and phenylalanine (8–9%) contents, some differences were present, notably in the relative content of lysine. The three fractions had identical amino acid sequences for the first ten residues at the N-terminal end. They also had identical sequences for the first five residues at the C-terminal end, with the exception that a mixture of two amino acids were released from position 4 of the 58,000 fraction only. Peptide mapping with three enzymes (trypsin, chymotrypsin and V8 protease) indicated that the 58 and 57,000 fractions were more closely related to each other than to the 54,000 fraction. It is suggested that the 57 and 58,000 fractions and the 54,000 fraction constitute two families of closely related polypeptides which are coded by genes derived from the duplication and divergence of a single ancestral gene.     

Closely related families of genes code for the alpha and alpha' subunits of the soybean 7S storage protein complex

Nineteen cloned cDNAs encoding the alpha and alpha'-subunits of the 7S seed storage protein in the soybean, Glycine max, have been isolated from a recombinant cDNA library constructed with mRNA from maturing seeds. In addition, a gene encoding an alpha'-subunit has been isolated from a recombinant Charon 4A phage library containing genomic Glycine max DNA. The cloned DNAs have been divided, on the basis of their endonuclease sites, into two main classes of sequences which differ in approximately 6% of their nucleotides. Whereas the proteins encoded within each DNA class are nearly identical, the proteins encoded by the two different classes of soybean DNAs are distinct and correspond to alpha and alpha'-subunits. Thus, the alpha and alpha'-subunits are coded for by two closely related multigene families. The amino acid differences in the portions of the alpha and alpha'-subunits presented in this paper occur primarily near the carboxyl-terminus. The 3' noncoding nucleotides of the cloned alpha and alpha'-subunit DNAs are more highly conserved than are the coding nucleotides. This conservation suggests that the 3' untranslated sequences of the alpha and alpha'-subunit mRNAs are functional in the expression of the alpha and alpha'-subunit proteins or in the stabilization of the 7S subunit mRNAs.