Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.     

A C-terminal sequence of soybean beta-conglycinin alpha' subunit acts as a vacuolar sorting determinant in seed cells

In maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension-cultured cells apart from seed cells. In this study, we examined the VSD of the alpha' subunit of beta-conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild-type alpha' was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid-like structures. Some of the wild-type alpha' was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C-terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The C-terminal region of alpha' was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that alpha' contains two VSDs: one is present in the C-terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C-terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.     

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an α subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.     

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.     

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.     

Proteome rebalancing in soybean seeds can be exploited to enhance foreign protein accumulation

Seeds possess a high intrinsic capacity for protein production that makes them a desirable bioreactor platform for the manufacture of transgenic products. One strategy to enhance foreign protein production involves exchanging the capacity to produce intrinsic proteins for the capacity to produce a high level of foreign proteins. Suppression of the alpha/alpha' subunit of beta-conglycinin storage protein synthesis in soybean has been shown previously to result in an increase in the accumulation of the glycinin storage protein, some of which is sequestered as proglycinin into de novo endoplasmic reticulum (ER)-derived protein bodies. The exchange of glycinin for conglycinin is quantitative, with the remodelled soybeans possessing a normal protein content with an altered proteome. The green fluorescent protein (GFP)-kdel reporter was transferred in a construct using the glycinin promoter and terminator to mimic glycinin gene expression. When expressed in soybean seeds, GFP-kdel accreted to form ER-derived protein bodies. The introgression of GFP-kdel into the alpha/alpha' subunit of the beta-conglycinin suppression background resulted in a fourfold enhancement of GFP-kdel accumulation to > 7% (w/w) of the total protein in soybean seeds. The resulting seeds accumulated a single population of ER membrane-bound protein bodies that contained both GFP-kdel and glycinin. Thus, the collateral proteome rebalancing that occurs with the suppression of intrinsic proteins in soybean can be exploited to produce an enhanced level of foreign proteins.     

Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon-intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the alpha'-subunit of beta-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or beta-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.     

Foreign gene products can be enhanced by introduction into low storage protein mutants

Transgenic rice expressing soybean glycinin in its endosperm was crossed with two types of low-glutelin mutants to determine how much storage the protein mutants can contribute to increases in glycinin accumulation. The glycinin level (102 microg/100 mg seed) in the parental transgenic line was enhanced to approximately 224-237 microg/100 mg seed within a genetic background deficient in glutelin (i.e. of low glutelins). The enrichment of this foreign gene product was compensated by a decrease in the expression of other endogenous prolamine and globulin storage proteins, resulting in an almost equivalent total amount of seed storage proteins. These results show that low storage protein mutants can provide potentially useful hosts for the expression of foreign genes, allowing a higher-level accumulation, because they can provide wider space for the accumulation of foreign gene products than in the normal host plant.     

Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors

Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.     

Electron microscopy of seed-storage globulins

The quaternary structures of a range of seed globulins, including examples of both the so-called 7 S and 11 S types, have been examined by electron microscopy. The legume 7 S proteins, phaseolin (bean), beta-conglycinin (soybean), and vicilin (pea), appear as flat discs of diameter ca. 8.5 nm and thickness ca. 3.5 nm formed by association of three subunit domains. Phaseolin converts to an 18 S tetramer at acid pH, and images recorded under these conditions suggest that four of the 7 S protomer discs associate to form the faces of a regular tetrahedron. The classical 11 S seed globulins, cucurbitin (pumpkin) and legumin (pea), are approximately spherical molecules of diameter ca. 8.8 nm composed of six subunits. In contrast, the hexameric 10 S storage protein from lupin seed, conglutin gamma, appears toroidal in shape with outer diameter ca. 10.3 nm and thickness ca. 2.2 nm. These results indicate that constraints imposed on seed proteins by their role in sustaining the germinating plant may have allowed a variety of different globulin structures to accumulate in the protein-storage bodies of seeds.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.     

Disappearance of immunoreactive glycinin and beta-conglycinin in the digestive tract of piglets

Soybean allergy represents a health threat to human and animals. Glycinin and beta-conglycinin, the main storage proteins in soybean, have been identified as major food/ feed allergens. The present study was conducted to investigate the disappearance of immunoreactive glycinin and beta-conglycinin in the digestive processes of piglets. Twelve crossbred piglets, weaned at 21 days of age, were allocated to three dietary treatments in a complete block design, each treatment with four replicates (female/male = 1:1). From day 22-28, the control group was fed diets without leguminous products, while the two treatment groups received diets containing 2.2% purified glycinin or beta-conglycinin. All piglets were slaughtered at 29 days of age and digesta was sampled from stomach, middle jejunum, caecum and colon. Results indicated that immunoreactive glycinin and beta-conglycinin decreased as the digesta descended down the digestive tract to 0.12% and 0.47%, respectively. Little immunoreactive glycinin was found in the digesta of caecum and colon, while immunoreactive beta-conglycinin was detected in the colon. Along the whole digestive tract the disappearance of immunoreactive glycinin was significantly higher than beta-conglycinin (p < 0.05).     

AtVPS29, a putative component of a retromer complex, is required for the efficient sorting of seed storage proteins

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) as larger precursors and are sorted to protein storage vacuoles, where they are converted into the mature forms. We report here an Arabidopsis mutant, maigo 1 (mag1), which abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag1 seeds mis-sort storage proteins by secreting them from cells. mag1 seeds have smaller protein storage vacuoles in the seeds than do wild-type seeds. The MAG1 gene encodes a homolog of the yeast (Saccharomyces cerevisiae) protein VPS29. VPS29 is a component of a retromer complex for recycling a vacuolar sorting receptor VPS10 from the pre-vacuolar compartment to the Golgi complex. Our findings suggest that MAG1/AtVPS29 protein is involved in recycling a plant receptor for the efficient sorting of seed storage proteins. The mag1 mutant exhibits a dwarf phenotype. A plant retromer complex plays a significant role in plant growth and development.     

Changes in growth,proteins and free amino acids of developing seed and pod of fenugreek

The growth and development under field conditions of the pods and seeds of two cvs of Trigonella foenum graecum are described. Samples were harvested at different stages of ripeness for the determination of dry matter, protein and free amino acid content. During maturation, reserves of solutes are established in the pod wall before the seeds begin their exponential phase of growth. Later, these reserves disappear, providing about 20% of the seed's requirements for nitrogen. SDS-electrophoresis was used to follow the formation of proteins and it was shown that the synthesis of storage proteins takes place prior to dehydration of the seed. Production soluble nitrogenous compounds precedes protein accumulation. Free amino acids follow the same pattern. 4-Hydroxyisoleucine represents nearly 80% of free amino acid of dry seeds. The concentration does not decrease in the later stages of maturation of the seed but this unusual amino acid is absent in the storage proteins of the seeds.     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

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 -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, -conglycinin (/ 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.