Characterization of a Small Sulphur-Rich Storage Albumin in Seeds of Alfalfa (Medicago sativa L.)

A 2S albumin fraction was characterized in seeds of alfalfa{^{Medicago sativa} L.). This low molecular weight (LMW) familyof disulphide-bonded proteins represents a major nitrogen andsulphur storage reserve for the alfalfa seed Characteristicof seed storage proteins, the 2S albumins are abundant in nitrogen-richglutarrune/glutamate/asparagine/aspartate (32%) In addition,this LMW fraction is high in cysteine (9%) and methionine (4%),amino acids which are under-represented in legume seed globulins.These 2S proteins start to accumulate during the early cotyledonstage of development, and are mobilized following germinationPulse-chase labelling experiments show that the 2S proteinsare synthesized as 'preproproteins', similar to 2S proteinsin other seeds. However, alfalfa 2S albumins are immunologicallyunrelated to these proteins. Key words: Seed development, sulphur-containing 2S storage protein, alfalfa (Medicago sativa)     

Isolation and characterization of cDNAs encoding oat 12S globulin mRNAs

Summary A cDNA library was made from poly(A⁺) RNA isolated from developing oat seeds, and oat globulin cDNA clones were identified by hybridization with synthetic oligonucleotides. Globulin clones were characterized by restriction enzyme mapping and cross-hybridization analysis. Based on these comparisons, four classes of globulin clones were distinguished. These clones hybridized to multiple DNA fragments in restriction enzyme digests of oat genomic DNA, indicating that the genes exist in a multigene family. The nucleotide sequence of one of the globulin cDNA clones was determined. The amino acid sequence derived from the DNA sequence verified its identity as an oat globulin and confirmed that the protein is synthesized as a precursor similar to legume 11S storage globulins. The basic polypeptide encoded at the 3 end of the mRNA was found to be homologous to the basic polypeptides of other 11S seed globulins.Abbreviations ds double stranded - kb kilobase Author to whom correspondence should be addressed. Journal paper number 10460 of the Purdue Agricultural Experimental Station.     

Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins

Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis.     

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.     

Immunochemical study of changes in reserve proteins of germinating soybean seeds

Changes in the reserve proteins of soybean seeds (Glycine max) were investigated by the techniques of disc electrophoresis and disc immunoelectrophoresis. Three different antisera were used in these studies, an anti-whole soybean extract serum 129, an anti-11S soybean protein monospecific serum 102, and an anti-7S soybean protein monospecific serum 132. At least 6 antigenically distinct components were found to be present in the proteins of the isolated soybean protein bodies. These components are metabolized at different rates during germination. The major soybean protein (11S component) is found to be present even after 16 days of germination, whereas the 7S component disappears after the ninth day. Histochemical observations of cotyledon sections during germination are also reported.     

A Comparison of Soybean Globulins and the Protein Bodies in the Protein Composition

The protein compositions between soybean globulins and the protein bodies were compared by gel filtration with Sephadex G-200, sedimentation analyses and disc isoelectric focusing.

From the results of the three comparisons, it was difficult to find an essential difference in the protein compositions of the both. And, the 7S and the 11S globulins were the main components in the both. This fact was more strongly suggested that these globulins were the typical reserve proteins.     

Identification of embryonal antigens of maize: Globulins as primary reserve proteins of the embryo

Normal and -irradiated caryopses of Zea mays L. were germinated, and the degradation of embryonal antigens (EA) was followed in the endosperms, scutella and embryonic axes of the seedlings, using double immunodiffusion, immunoelectrophoresis and quantitative immunoprecipitation. The predominant transient EA were presumed to be storage proteins related to the reserve globulins of dicotyledonous seeds. Therefore globulins were isolated from maize scutella, purified by (NH₄)₂SO₄ fractionation and isoelectric precipitation, and the molecular weights of the polypeptide units were estimated by discontinuous sodium-dodecyl-sulphate slab electrophoresis. The globulins were found to be identical with the predominant EA and amounted to about 40% of the protein nitrogen in the embryos of mature, non-germinated caryopses. The presumed reserve function of the globulins and the characteristic time course of their degradation in embryonic axes and scutella of maize seedlings are discussed in relation to the two-step pattern of mobilization of nitrogen reserves in germinating cereal caryopses.Abbreviations AG, AR and AS antisera to globulins and to root and scutellum proteins, respectively - CA common antigens - DAP days after pollination - EA embryonal antigens - ME 2-mercaptoethanol - SDS-PAGE discontinuous sodium-dodecylsulphate slab polyacrylamide-gel electrophoresis - TCA trichloroacetic acid     

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.     

Characterization of Seed Protein Fractions from Castanea spp

Seed proteins of Castanea sativa and C. crenata were extractedby sequential procedures. Albumins accounted for over 20% ofthe total protein. Globulins were the main storage proteinsand represented close to half of the total protein. Additionof 2-mercaptoethanol was essential for maximum extraction ofthe globulins, a fraction with an amino acid composition similarto that of the 1 IS storage protein from legumes. Negligibleamounts of prolamins (<3%) were present in the seeds. Glutelinsrepresented just under 30% of the total protein. The proteinfractions were characterized by electrophoresis in polyacrylamidegels. The glutelin fraction contained unextracted globulinseven when 2-mercaptoethanol was used. Key words: Castanea spp, seeds, storage protein     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and β-glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Seed globulins of various species of cucurbitaceae

Globulins isolated from 6 species of the Cucurbitaceae family (C. maxima, C. pepo, C. moschata, Luffa cylindrica, Lagenaria vulgaris, and Momordica charantia) were studied. Nitrogen content of the globulins varied from 18.3 to 18.8%, with a mean of 18.6%. Of the individual amino acids, the most abundant were arginine and glutamic acid. Content of histidine, proline, serine, and tyrosine showed relatively higher variability within the group of species compared. MWs of the globulins, determined with Sephadex G 200, were 241 000 (C. maxima, C. pepo), 248 000 (C. moschata, L. cylindrica), 256 000 (L. vulgaris), and 218 000 (M. charantia). By ultracentrifugal analysis of globulins in 2 M NACl (pH 8), 3 fractions were identified in all the species except M. charantia, their sedimentation coefficients being in the range of (1) 5.2 S–7.2 S, (2) 15.3 S–17.2 S, and (3) 10.4–11.2 S. The latter fraction predominated in all the species, its amount was 94–96% of total globulins. Differences in electrophoretic properties of the globulins and their subunits produced in the presence of 8 M urea and by oxidative splitting with performic acid, respectively, and results of electrophoresis in SDS-acrylamide gels are also discussed.     

7S Globulins from Phaseolus vulgaris L.: Impact of Structural Aspects on the Nutritional Quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS–PAGE showed major bands corresponding to the phaseolin subunits (43–53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 °C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of β-sheet and β-turn structures, together with a contribution at 1687 cm^?1 that was assigned to intramolecular β-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Identification of the 7S globulin with β-conglycinin in soybean seeds

The 7S globulin, a major ultracentrifugal component with the 11S globulin, was identical with β-conglycinin one of four antigenic components in the reserve proteins of soybean seeds (Glycine max). Double gel immunodiffusion and immunoelectrophoresis in agar gel were used for their identification. In addition, some characteristic properties on ultracentrifugation and in carbohydrate content agreed well between the proteins. Their MWs were ca 180000.     

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.     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [³⁵S]sulfate into protein. Incorporation of [³⁵S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the α and β globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

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     

Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post-mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran-polyethylene glycol two-phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran-polyethylene glycol two-phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP-glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS-PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two-phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)-binding proteins in aleurone.     

Precursors of the Cocoa-Specific Aroma Components are Derived from the Vicilin-Class (7S) Globulin of the Cocoa Seeds by Proteolytic Processing

Essential precursors of the cocoa-specific aroma components are formed during fermentation of cocoa seeds in the tropics. During the past decades, indications have accumulated that these aroma precursors are derived from seed proteins by acid-induced proteolysis. This has been recently corroborated by in vitro studies on the formation of the aroma-related components. It has been shown that the essential proteolysis products are derived from the vicilin-class (7S) globulins of the cocoa seeds by the cooperative action of an endogenous aspartic endoprotease and a carboxypeptidase.