7S Storage Proteins Research Articles

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae.

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds.

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a 'hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.

Seed storage proteins in cultivars and subspecies of alfalfa (Medicago sativa L.)

AbstractSeed storage proteins were analysed in 27 varieties of alfalfa (Medicago sativaL.); these included five subspecies (glomerata, caerulea, falcata, hemicycla, praefalcata), seven of the nine sources of Medicago germplasm introduced into North America and a sample of additional cultivars. The protein patterns were remarkably consistent for all of these taxa. One-dimensional and two-dimensional gel electrophoresis revealed only minor differences in polypeptide composition within each of the three major classes of storage protein (7S globulin, 11S globulin, 2S albumin). The slight variations that were found provided no information on either parentage or evolutionary relationships amongst these particular taxa. Nonetheless, persistent and reproducible heterogeneity of some minor polypeptides of 11S globulin (medicagin) may be useful under other circumstances for cultivar identification in alfalfa. Both subfamilies (I and II) of the 11S globulin were strongly expressed in all of the cultivars and subspecies examined. It was concluded that this structural divergence within the 11S storage protein family predated the evolution of the M. sativa L. species complex. Most of the variability in storage proteins was quantitative. However, even this variability was reduced when data were standardized with respect to seed dry weights. The consistent similarities in qualitative and quantitative expression of seed storage proteins amongst all of these taxa suggest a high degree of uniformity in both seed physiology and genetics within the alfalfa species complex.

Changes in interfacial behaviour, emulsifying and foaming properties of faba bean legumin after modification with dimethylsuberimidate

The effect of a rising rigidity and surface hydrophobicity of the 11S storage protein from faba beans--legumin--induced by chemical modification with dimethylsuberimidate (DMS) on some surface functional properties was studied. Short-time adsorption kinetics using a droplet-volume tensiometer, pressure transformation and desorption behaviour of monolayer using a film balance, and emulsifying and foaming properties were determined to characterize surface activity and interfacial film forming behaviour. Tensio-active properties at the air-water interface, i.e. decay in surface tension and pressure transformation in monolayer, were improved by modification. However, a decrease in emulsifying activity, foam capacity and foam expansion after modification of the legumin points to an overall deterioration of energy-induced film forming behaviour. The results support the view that surface activity is generally governed more by molecular flexibility than by surface hydrophobicity.

Coffee seeds contain 11S storage proteins

A legumin‐like seed protein was purified from the endosperm of coffee (Coffea arabica L. cv. Colombia). In contrast to legumes, where efficient storage globulin extraction requires buffered saline solutions well above the acidic pKI of the globulins, coffee legumin is readily extracted with acidic aqueous buffers. The coffee legumin migrates like other 11S storage globulins in sucrose gradients. Subunits of coffee legumin have an apparent molecular mass of about 55 kDa after one‐dimensional SDS‐polyacrylamide gel electrophoresis in the absence of a reducing agent. In the presence of 2‐mercaptoethanol, two polypeptides appear that have apparent molecular masses of 33 and 24 kDa. Two full‐length cDNAs were generated from mRNA of developing seeds that were more than 98% homologous. They had open reading frames of 1 458 and 1 467 bp. Each encoded legumin precursors of 486 and 489 amino acids, respectively (Mr=54 136 and 54 818). Examination of a 5′ promoter region from a coffee legumin gene revealed a putative legumin‐box. Genomic DNA from C. arabica was digested with six different restriction endonucleases. After separation of the fragments by electrophoresis, single discrete fragments on DNA blots hybridized strongly to a cDNA probe for the acidic chain. Other fragments that hybridized weakly with this probe were visible after hybridization at very low stringency. DNA from other species and commercially important cultivars that comprise the genus Coffea produced similar results. Immunocytochemical studies revealed that some legumin was detected in the cytoplasm in mature coffee seeds, but that the majority of it was in large storage vacuoles that accounted for most of the cell volume.

Ultrastructure and immunolabelling of fungi cells treated with Vigna unguiculata vicilins (7S storage proteins)

Scanning electron microscopy showed that vicilins from cowpea seeds affect the growth and inhibit spore germination of Fusarium oxysporum and leads to abnormal development (sporulation) of Saccharomyces cerevisiae cells. Control treatments induced normal hyphal and cell development. Fluorescence microscopy and immunolabelling analysis showed that anti-vicilin serum strongly labelled the cell surfaces of yeast and filamentous fungi, previously incubated with these seed proteins. These results suggest that the inhibitory effects of cowpea vicilins on fungi growth may be exerted through their ability to bind to the cell surfaces of the micro-organisms, cell wall and/or plasma membrane.

Effect of salts on soy storage proteins defatted with supercritical CO2 and alcohols

AbstractThe primary objective was to determine whether salts will stabilize soy storage proteins against the denaturing effects of alcohols or the heat and pressure used in supercritical CO2 during the defatting process. Nitrogen solubility index (NSI) and differential scanning calorimetry (DSC) were used to monitor the denaturation of proteins. A variety of salt solutions used to hydrate full‐fat soy grits increased the thermal stability of both 7S and 11S storage proteins. DSC was used to monitor their denaturation temperature. Neutral salt hydrations followed the lyotropic series for protein stabilization. Of the salts evaluated, the test results indicate that the reducing salt, sodium sulfite, and the neutral salt, sodium sulfate, when used to steep beans, yielded significantly higher NSI than did the water‐steeped controls or other salt treatments after partial defatting with absolute isopropanol or ethanol and supercritical CO2. However, these same salt treatments did not as effectively stabilize the proteins against the denaturing effects of ethanol more aqueous than 84% when these alcohols were used as the defatting medium.

Sequence and expression of embryogenesis-specific cDNAs encoding 2S seed storage proteins in Pseudotsuga menziesii [Mirb.] Franco.

Differential screening of a cDNA library constructed from polyadenylated RNA from developing Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seeds resulted in the isolation of four full-length cDNA clones (PM2S1, PM2S2, PM2S3 and PM2S4) encoding isoforms of 2S seed storage proteins. The deduced amino acid sequences had low similarity to the angiosperm 2S storage proteins such as 2S albumin, napin and alpha-amylase/trypsin inhibitor yet contained all conserved cysteines in an arrangement suggestive of a structural similarity between the 2S seed storage proteins from gymnosperms and angiosperms. Southern blot analysis of genomic DNA suggested that the Douglas-fir 2S seed protein genes consisted of at least two sub-families, each including several gene members. Northern blot analysis revealed that all four PM2S mRNAs were present specifically in seeds and temporally during seed development. However, the decline in PM2S2 mRNAs in the megagametophytes occurred before that of the other mRNAs, suggesting that their gene expression was regulated differentially. The accumulation of PM2S transcripts in the haploid megagametophytes started during early embryogenesis and reached a peak before that in diploid zygotic embryos. The mRNAs for PM2S were present in Douglas-fir somatic embryos at the same developmental stages as those in the zygotic embryos, and abscisic acid and osmoticum stress were necessary for the expression of PM2S genes in somatic embryos.

Legume vicilins (7S storage globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells

Vicilin (7S storage proteins) isolated from different legume seeds were shown to inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied with the origin of vicilins. It was more than 90% for vicilins from cowpea ( Vigna unguiculata, cultivar pitiuba) and equal to 65% for vicilins from Vigna radiata, in the case of Saccharomyces cerevisae. Vicilins from cowpea seeds inhibited the glucose stimulated acidification of the medium by S. cerevisae up to 60%. We have also observed that vicilins bind to yeast cells. We suggest that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H + pumping, cell growth and spore formation. A final consequence of the yeast growth inhibition by vicilins is (probably) the formation of spores.

Legume seed vicilins (7S storage proteins) interfere with the development of the cowpea weevil (Callosobruchus maculatus (F))

Vicilins (7S storage proteins) isolated from the seeds of the legumes Vigna unguiculata (cowpea), Vigna angularis (adzuki bean), Canavalia ensiformis (jack bean), Glycine max (soybean), Phaseolus vulgaris (common bean) and Phaseolus lunatus (lima bean) were shown to be immunologically related and to bind to a chitin matrix. The effect of the isolated vicilins on the development of the cowpea weevil Callosobruchus maculatus was examined. Vicilins from all non-host seeds, including those of the C maculatus-resistant cowpea line IT81D-1045, strongly inhibited larval development (ED 50 of 1.07 ± 0.32% to 2.22 ± 0.64%). Vicilins from the C maculatus-susceptible cowpea CE-31 and adzuki bean seeds were the exception with ED 50 of 6.25 ± 0.75% and 5.40 ± 1.54%, respectively. These results correlate well with the host range of C maculatus and are possibly a reflection of the low digestibility of vicilins by insect midgut proteinases in addition to the ability they show in binding to chitin-containing structures like the ones found in the bruchid midguts.

Seed-specific regulation of the napin promoter in Brassica napus

Summary The expression of the 2S storage protein napin in Brassica napus is dependent upon ABI3. Mutation analyses identified a promoter segment between −152 to −120, denoted the B-box, to be critical for the activity of the promoter in transgenic tobacco seeds. The B-box, which is highly conserved in all 2S promoters and which displays similarity to abscisic acid response elements, binds at least two different nuclear protein complexes. Mutation of either of the two B-box elements binding nuclear protein complexes abolished the activity of the promoter in transgenic tobacco seeds. The concatemerised B-box in front of the minimal 35 S promoter from cauliflower mosaic virus, directed seed-specific transcription in transgenic tobacco and was able to induce transcription in young tobacco seedlings ectopically expressing ABI3. This implies that the B-box is one region through which ABI3 controls the napin promoter.

Molecular cloning and expression patterns of Cu/Zn-superoxide dismutases in developing soybean seeds.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development.

Previous studies have shown that recessive mutations at the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON1 (LEC1) loci lead to various abnormalities during mid-embryogenesis and late embryogenesis. In this study, we investigated whether these loci act in independent regulatory pathways or interact in controlling certain facets of seed development. Several developmental responses were quantified in abi3, fus3, and lec1 single mutants as well as in double mutants combining either the weak abi3-1 or the severe abi3-4 mutations with either fus3 or lec1 mutations. Our data indicate that ABI3 interacts genetically with both FUS3 and LEC1 in controlling each of the elementary processes analyzed, namely, accumulation of chlorophyll and anthocyanins, sensitivity to abscisic acid, and expression of individual members of the 12S storage protein gene family. In addition, both FUS3 and LEC1 regulate positively the abundance of the ABI3 protein in the seed. These results suggest that in contrast to previous models, the ABI3, FUS3, and LEC1 genes act synergistically to control multiple elementary processes during seed development.

Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour

Background Seed proteins have been found to cause hypersensitivity by ingestion or inhalation. Rapeseed fiour was responsible for allergic symptoms in a patient, who develops into allergy to mustard spice. Objective To determine the presence of allergenic proteins in rapeseed fiour, and analyse the structure of the main component and its crossreactivity with the mustard allergeti. Methods SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequent immunoblotting with a serum from a rapeseed allergic patient were performed to detect IgE-binding proteins. Proteolytic digestiotis and high performance liquid chromatography were used to obtain the peptides from the allergenic BnIII napin from rapeseed flour. Automatic Edman degradations were carried out to determine their amino acid sequences, which were compared with other sequences in nucleotide and amino acid sequence databases. Crossreactivity assays were carried out by ELISA inhibition using sera from a rapeseed allergic patient and from patients allergic to mustard. Results The 2S albumins of rapeseed were recognized by the serum from a patient allergic to this seed. The most abundant isoform of the allergenic napins, Bnlll, was used for structural and immunological analysis. The protein consists of two different chains of 9.5 and 4,5 kDa. Their complete amino acid sequences were determined. The protein exhibited structural relationships with other napin-like storage proteins from seeds. IgE and IgG crossreactivity between rapeseed and mustard allergens was also demonstrated. Considering the structural and immunological data, certain polypeptide regions are suggested to be involved in the allergenicity of these proteins. Conclusions Rapeseed contains 2S storage proteins which may cause allergy in hypersensitive individuals. These proteins exhibit great sequence similarity with 2S albumins from different seeds. Crossreactivity between mustard and rapeseed flours can be explained by sequetice homology.

Characterization of 2S and 7S storage proteins in embryos of oil palm

Storage proteins that accumulated during oil palm embryo development were extracted, purified and characterized. Only water- and low-salt-soluble proteins, with respective sedimentation coefficients of 2S and 7S, were detected in mature embryos. After purification by gel filtration, the various protein classes identified were characterized by electrophoresis and amino acid composition analysis. The 2S proteins comprise polypeptides of 22 kD and 19 kD, which are acidic (pI < 6) and basic (pI > 9) respectively. The 7S proteins predominate and are heterogeneous oligomers (Mr of 156 and 201 kD), comprising a polypeptide triplet of Mr between 45 and 65 kD with no disulphide bonds. Their amino acid composition is broadly similar to those of the 7S proteins of other monocotyledon embryos, but differs from those of the legume 7S vicilins. Histological examinations and electrophoresis showed that the 2S and 7S proteins appeared at the third month after fertilization, and no qualitative changes were detected up to the sixth month of embryo development. The characterization of the embryo storage proteins of oil palm is discussed with reference to legume, cereal and palm seed storage proteins. This study will enable further investigation of storage protein synthesis during somatic embryogenesis.

Structural comparison of the precursor and the mature form of napin, the 2S storage protein in Brassica napus.

The 2S storage protein napin from Brassica napus var. L. is synthesised as a precursor protein at the endoplasmic reticulum and transported along a gradient of decreasing pH to the vacuole, where two propeptides are removed to produce mature napin. The structures of pronapin expressed in insect cells and mature napin from rape seed were characterised. Limited proteolysis with several endoproteases cleaved primarily in the propeptides, suggesting that the propeptides are exposed to the exterior of the protein. Immunological comparison in parallel with circular dichroic spectrometry, both at neutral and acid pH, indicated that the propeptides had only a minor influence on the conformation of the regions of the molecule that correspond to mature napin.

Production and characterization of monoclonal antibodies against 11S storage protein from pea seeds

Antibodies can be used as probes to investigate the structure of 11S storage proteins, their subunit composition and structural modifications induced by technological treatments. Monoclonal antibodies have been raised against Pisum sativum legumin (11S storage protein). Their binding characteristics were examined by direct, sandwich and competitive ELISA and by immunoblotting against legumin and 11S type storage proteins from other species. One of the MAbs, reacting with all 11S proteins tested, recognizes a discontinuous epitope accessible on the surface of the native hexameric protein but destroyed in dissociated legumin. Two antibodies recognize sequential epitopes belonging to a region of the acidic polypeptides present on the surface of the native legumin. These two MAbs cross-react only with pea and bean 11S proteins. Two other MAbs are specific for sequential epitopes buried in the native protein, localized, respectively, on acidic and basic polypeptides. The MAb reacting with the acidic polypeptide exhibits very specific binding for pea legumin. By contrast, the MAb specific to the basic polypeptide cross reacts with 11S proteins studied. This work shows the potential of this approach for the characterization of the conformation of the 11S proteins and the investigation of structural modifications.

Embryos of alfalfa (Medicago sativa L.) can synthesize storage proteins after the completion of germination, in response to abscisic acid and osmoticum, if maturation drying is prevented

The major 2S, 7S and 11S storage proteins are synthesized at the midto late stages during development of alfalfa seeds, mainly within the embryos. Mature dry seeds cannot synthesize these proteins upon subsequent germination and growth. When embryos were isolated from the seed during development (stages VII and VIII) and placed on water or nutrient medium, they germinated. They exhibited a pattern of protein synthesis which was identifiable as germinative/post-germinative, and was identical to that pattern synthesized by the embryos of germinated dry seeds, and of embryos from seeds that were subjected to drying prematurely at stages VII and VIII. When, after 48 h from the start of imbibition, abscisic acid or osmoticum was introduced to germinated embryos which were isolated at stage VII and not desiccated, the synthesis the 11S storage protein was restored to a level comparable to that of stage VII embryos before isolation. This was accompanied by an increase in transcription of the 11S storage-protein gene, even at a time when storage-protein mobilization was occurring. Thus storage-protein synthesis is not terminated temporally during alfalfa seed development, but is maintained well beyond the time of physiological maturity if the embryo is maintained under conditions which are favorable to development. Transfer of germinated stage VII embryos, which had first been desiccated, to abscisic acid or osmoticum 48 h after imbibition started did not result in any restoration of storage-protein synthesis. Thus desiccation, either prematurely or at maturation, causes storageprotein synthesis to cease and within the embryos this process is no longer responsive to abscisic acid or osmoticum.

Effect of abscisic acid, osmoticum and desiccation on 11S storage protein gene expression in somatic embryos of white spruce

The effect of ABA, non-permeating osmoticum and late stage maturation drying on 11S storage protein gene expression in white spruce somatic embryos was investigated. A partial cDNA clone encoding the β-polypeptide of white spruce 11S legumin-like seed storage protein (named β-coniferin) was employed as a probe. Northern blot analyses demonstrated that in somatic embryos matured with 16 μM ABA and 7.5% PEG, the levels and patterns of accumulation of transcripts during early and mid-stages of embryogenesis were comparable to those observed in zygotic embryos at a similar morphological stage. Steady-state levels of coniferin transcripts declined when late stage somatic embryos were exposed to a desiccation treatment. Levels of coniferin transcripts in somatic embryos matured with 16 μM ABA and low osmoticum were substantially lower than those of somatic embryos matured with 7.5% PEG and 16 μM ABA. Increasing concentrations of ABA resulted in higher levels of accumulation of coniferin transcripts, however, the levels in somatic embryos matured with 32 μM ABA and low osmoticum remained lower than those of somatic embryos matured on 16 μM and 7.5% PEG indicating that the latter treatment is optimum for induction and accumulation of coniferin transcripts. The similarity of 11S storage protein gene expression in somatic embryos with zygotic embryos, along with their sensitivity to ABA and osmoticum indicates that somatic embryos may provide a useful system to facilitate studies of gene regulation in conifers.