Comparative study of seed albumins in the Old-WorldLupinus species (Fabaceae) by reversed-phase HPLC

Seed albumins and 2S proteins isolated from the albumin fraction of 36 accessions representing 10 Old-WorldLupinus species (5 smooth- and 5 rough-seeded) were studied using reversed-phase high-performance liquid chromatography. In addition, the globulin fraction was analyzed to determine its 2S protein content. The performed separations showed the suitability of RP-HPLC technique in the analysis of variation of the seed albumin composition in lupins. In the group of rough-seeded lupins, 3 types of RP-HPLC elution profiles of albumins were distinguished: (1)L. atlanticus, (2)L. cosentinii andL. digitatus, (3)L. palaestinus andL. pilosus. All the species of this group were found to have proteins not observed in smooth-seeded species. Smooth-seeded species exhibited more abundant protein spectra, each species distinguishing by its specific RP-HPLC elution profile. It was found that 2S proteins classified as 2S albumins were responsible for the observed variation. Depending onLupinus species, the 2S albumin class consists of two to six proteins.     

Immunoelectrophoretic analysis of seed proteins from Pisum sativum L.

Summary Soluble proteins of pea seed were investigated by quantitative immunological methods. Vicilin, legumin, pea seed lectin (PEA), 26 albumins and a globulin (B₁) were detected and observed during seed development, germination and under different extraction and fractionation procedures. Vicilin and legumin were found to be immunologically distinctly different. Legumin was found to be comprised of two similar proteins, Legumin species I and II. Vicilin, but no legumin, was detected in the embryonic axis.Three albumins, B₁ and PEA were found to be synthesized after the onset of legumin synthesis.Among the pea lines investigated, one line exhibited distinct differences with respect to the albumins and PEA.Some observations indicate that PEA might interact with other seed proteins of pea.     

Electrophoretic patterns of seed albumins in the Old-WorldLupinus species (Fabaceae): Variation in the 2S albumin class

PAGE and SDS-PAGE electrophoretic analysis of 31 accessions of 10 Old-WorldLupinus species, 5 smooth-seeded and 5 rough-seeded, covered total seed albumins and 2S albumins isolated by a solid-phase extraction. PAGE albumin patterns showed a distinct difference between the smooth- and rough-seeded lupins. SDS-PAGE analysis of seed albumins revealed interspecific differences, mainly due to the 2S albumins. The differences were especially marked in the smooth-seeded species. In the rough-seeded lupins the following subgroups were distinguished: (1)L. atlanticus, (2)L. cosentinii andL. digitatus, (3)L. palaestinus andL. pilosus. Evidence was provided that the 2S albumin class contains conglutin , so far classified as a globulin. The results are discussed with reference to taxonomic relationships of the Old-World lupins and characterization of the lupin seed albumin fraction.     

Evidence for a precursor molecule of Brazil nut 2 S seed proteins from biosynthesis and cDNA analysis

Summary Seed storage proteins were extracted from Brazil nut (Bertholletia excelsa H.B.K.) seed embryos at various maturation stages. Salt-soluble and water-soluble proteins (globulins and albumins) were separated by gel chromatography and exhaustive dialysis against water. Both fractions were analysed by one- and two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis revealed that both fractions are unusually high in methionine. The albumins consist of a family of low molecular weight polypeptides that are heterogeneous with respect to pI and are identical to the high methionine 2 S proteins described by Youle and Huang (1981). The biosynthesis of this class of proteins in maturing embryos was followed by in vivo labelling combined with immunological studies. Western blotting with monospecific antibodies against purified 2 S albumins and sequencing of a nearly complete cDNA clone revealed that they are synthesized via a precursor polypeptide.     

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)     

Albumin storage proteins in the protein bodies of castor bean

Of the total protein in the protein bodies of castor bean (Ricinus communis L.), approximately 40% is represented by a group of closely related albumins localized in the matrix of the organelle. This group of albumins has a sedimentation value of 2S and is resolved into several proteins of molecular weight around 12,000 daltons by sodium dodecyl sulfate-acrylamide gel electrophoresis. It has a high content of glutamate/glutamine and undergoes rapid degradation during the early stage of germination. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins, in addition to globulins, glutelins, and prolamines, are important storage proteins in seeds.     

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.     

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

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

Purification and Characterization of Proteins from the 2S Fraction from Seeds of the Brassicaceae Family

The 2S protein fractions from Brassica rapa, Brassica oleracea,and Brassica napus seeds have been obtained and their componentspurified and characterized. These albumins represent about 13%of the total seed protein extracted with saline buffer. Thecalculated molecular weights of the eleven purified proteinsare very similar, about 14 500 Daltons, although two componentsisolated from B. napus exhibit a lower molecular weight. Theamino acid compositions of the isolated proteins present a seriesof common features: a high content of Cys and basic residuesas well as a very high amount of Pro (15%) and Glx (30%). Theeleven purified proteins cross-react with antibodies againstSin a I, the 2S protein from yellow mustard seeds. The obtainedresults suggest the existence of homology between the 2S albuminsof Brassicaceae seeds. Key words: Brassicaceae, seeds, storage protein     

Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants

We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein.     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同,种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中,醇溶蛋白在粗面内质网合成后形成蛋白质聚集体,直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后,根据各自的分选信号进入特定的运输囊泡,经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中,然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同, 种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中, 醇溶蛋白在粗面内质网合成后形成蛋白质聚集体, 直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后, 根据各自的分选信号进入特定的运输囊泡, 经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中, 然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

Plant food allergens--structural and functional aspects of allergenicity

The three dominating plant food allergen groups belong to the prolamin and cupin superfamilies and to the family 10 of pathogenesis-related proteins. The prolamin superfamily comprises allergenic 2S albumins, nonspecific lipid transfer proteins and cereal alpha-amylase/trypsin inhibitors. These allergens have related structures and are stable to thermal processing and proteolysis. The cupin superfamily comprises the allergenic 7S and 11S globulin storage proteins from peanuts, soybean and tree nuts which are heat stable and can form immunogenicity enhancing aggregates. The Bet v 1 family of allergens includes tree pollinosis-associated food allergens with low stability which induce the symptoms of the oral allergy syndrome.     

Fractionation and Electrophoretic Patterns of Storage Proteins of Ebenus cretica. A preliminary Survey as a Tool in Taxonomy

Seed storage proteins of Ebenus cretica were fractionated to albumins, globulins, prolamins and glutelins according to their solubility in water, 0.5 M NaCl solution, 55 % propanol-2 and 0.125 M sodium borate (pH 9.0) containing 0.5 % SDS (sodium dodecyl sulfate) solution, respectively. Glutelins consist of the major (about 81 %) fraction of the total extracted proteins. Analysis by SDS-PAGE revealed that the total extracted protein patterns from different racemes of the same plant were similar, while those from seeds of different plants were different. In addition, distinct differences were observed within protein patterns of alkaline extractable glutelin fractions and salt soluble globulin fractions. In E. cretica four ecotypes (A – D) were distinguished by SDS-PAGE of total extracted seed proteins. The last method was more simple and rapid than others and was suggested for screening analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical genetics of a 7S globulin-like protein from Pinus pinaster seed

The megagametophytes of seeds of Pinus pinaster Ait. contain two types of oligomeric globulins of approximately 175 and 190 kDa that are comprised of 47-kDa and 27- and 22-kDa, monomers, respectively, joined by weak interactions. The 27- and 22-kDa components were purified and their N-terminal sequences determined. Both polypeptides were inherited as if they were coded by a single unit of recombination. The results obtained suggest that these two polypeptides originate from a single protein that undergoes proteolytic processing. The characteristics of this P. pinaster globulin indicate that it is a member of the 7S globulin family of seed storage proteins.     

Identification and Characterization of the Seed Storage Proteins from Alfalfa (Medicago sativa)

The major seed storage proteins in alfalfa are medicagin (alegumin-like globulin), alfin (a vicilin-like globulin) anda family of Lower Molecular Weight albumins (LMW₁₃). These comprise30%, 10% and 20%, respectively, of the total extractable proteinfrom cotyledons of mature seeds. Alfin is a heterogeneous oligomericprotein (Mr 150 kD) composed of polypeptides ranging in sizefrom Mr 50 to 14 kD (₁,-₆; 50, 38, 32, 20, 16 and 14 kD, respectively).Medicagin is also a high molecular weight oligomeric protein,but requires high concentrations of salt for solubilization.It is comprised of a family of individually distinct subunits,each composed of an acidic polypeptide (A₁–A₉; Mr 49 to39 kD) linked via disulphide bond(s) to a basic polypeptide(B₁, B₂, B₃; Mr 24, 23 and 20 kD, respectively). This pairingis highly specific and two families are recognizable on thebasis of the B polypeptide (B₃ or B₁/B₂). Subunits (M_r 50–65kD) are assembled as trimers (8S) or larger oligomers (12S–15S)in mature seeds. The lower molecular weight albumins (LMW₁–₃)are acidic (pl<6), and consist of sets of disulphide-bondedpolypeptides (Mr 15 and 11 kD). Key words: Medicago sativa, seed storage proteins, alfin, medicagin     

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.     

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.