Electrophoretic analysis of seed proteins in the dicotyledoneae

SDS-PAGE was used to analyze seed protein extracts from plants representing 58 orders of the Dicotyledoneae. In most seeds, includingMagnolia grandiflora, the most prevalent storage protein class appeared to be the 11 S or legumin-like proteins. These data suggest that the legumin-like proteins are ubiquitous throughout the plant kingdom. Supported by the Mississippi Agricultural and Forestry Experiment Station, Publication Number J-7788.     

Characterization of olive seed storage proteins

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

Secondary structure prediction of seed storage proteins

The comparison of partial primary structure of seed storage proteins leads to show homologies inside of each considered family (Legume seed legumins and cereal prolamins). Predicted secondary structures deduced from the presently known sequences also exhibit considerable homologies, which implies a severe conservatism of these proteins. Short repetitive segments of sequence of 5-20 residues are frequently occurring and give rise to the prediction either of beta-structure (or alpha-helix) bonded by beta-turns or of successive beta-turns. The latter conformation, which would be able to form a helicoidal arrangement, could contribute to a maximal packing of the protein molecules inside of the subcellular organelles (protein bodies) within which they are confined. As the only known function of seed storage proteins is to provide amino acids to the embryo, it is suggested that their ability to occupy a minimal volume is actually a reasonable explanation of their extreme conservatism in the course of evolution.     

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.     

Assembly and transport of seed storage proteins

Plant seeds store nitrogen by accumulating storage proteins in protein bodies within various compartments of the endomembrane system. The prolamin storage proteins of some cereal species are normally retained and assembled into protein bodies within the ER. Yet, these proteins lack a C-terminal KDEL/HDEL signal, suggesting that their retention is regulated by novel mechanisms. Furthermore, in other cereal species, such protein bodies formed within the ER may be subsequently internalized into vacuoles by a special route that does not utilize the Golgi complex. Thus, studies of the routing of seed storage proteins are revealing novel mechanisms of protein assembly and transport in the endomembrane system.     

Immunochemical studies on barley seed storage proteins

The antigenic relationships between the prolamins of barley, rye and wheat have been studied by examining the specificity of an antibody to C hordein in a quantitative study using a laser nephelometer. The antibody reacts weakly with B hordein and strongly with 75-kdalton and 40-kdalton -secalins from rye and ₃ some -gliadins from wheat. Absorption experiments and immunodiffusion tests indicate that there are shared antigenic determinants for most of the prolamins. All the species with reacting prolamins belong to the sub-family Festucoideae of the Gramineae. The prolamins of maize, pearl millet and sorghum, species of the sub-family Panicoideae, do not react. The results confirm the known lack of homology between the prolamins of the two sub-families and also indicate the presence of relationships not yet established between C hordein, the 75-kdalton and 40-kdalton -secalins and also ₃ gliadin.Abbreviations HMW high molecular weight - PAGE polyacnylamide-gel electrophoresis - PBS phosphate-buffered saline - RLS relative light scattering - SDS sodlum dodecyl sulphate     

Electrophoretic seed globulin patterns and species relationships in the genus Lens Miller

SDS-PAGE analysis of seed globulins covered 200 accessions of the following Lens taxa: L. culinaris subsp. culinaris, L. culinaris subsp. orientalis, L. odemensis, L. ervoides, L. nigricans, L. lamottei and L. tomentosus. The number of polypeptide bands detected in particular taxa varied from 22 in L. lamottei to 35 in L. ervoides. All the taxa under study showed variation due to differences among accessions and individual variation. Electrophoretic data were subjected to statistical analysis by the UPGMA method based on Euclidean distances. In the case of L. culinaris subsp. culinaris, some distinctness of the microsperma accessions from southern and central Asia was found. As to relationships among the studied taxa, the obtained results showed that L. culinaris subsp. culinaris appeared to be most closely allied to L. odemensis, while L. culinaris subsp. orientalis was found to be the closest relative of L. tomentosus. The four taxa formed one cluster separated from L. lamottei and L. ervoides. L. nigricans was shown to be the most divergent taxon.     

Electrophoretic characterization of Amaranthus L. seed proteins and its systematic implications

The seed protein profiles of 11 Amaranthus taxa (Amaranthaceae) from Spain were studied. These profiles were evaluated as a chemical character to clarify the taxonomic complexity in the genus. Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of the Amaranthus seed proteins studied showed a range of peptides varying from 64 to 12 kDa, with a larger number of protein bands observed between 25.1 and 12 kDa. For the taxonomic study, 14 bands, some of them subdivided into several isoforms, were considered. The similarity analysis based on the SDS-PAGE profile is a useful character for the discrimination of species in Amaranthus , except for A. cruentus and A. hypochondriacus , for which a hybrid population was found. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 57–63.     

Immunological relationships among the major seed proteins of cereals

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

Genetic relationships in the genus Cicer L. as revealed by polyacrylamide gel electrophoresis of seed storage proteins

Summary Total seed storage protein of the cultivated chickpea, C. arietinum L., and eight other wild annual Cicer species (all 2n = 16) was separated and compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The seed-protein profile was a conservative and species-specific trait. Relative interspecific similarities of protein patterns were estimated using Jaccard's similarity index, and a cluster analysis was performed. The resultant dendrogram generally agreed with the limited data already available on interspecific relationships in Cicer based on morphological characteristics, crossability, genome pairing in hybrids, karyotypes and isozyme analysis. The difference between the profiles of C. judaicum and C. pinnatifidum supported the idea that they are indeed two separate species. The closest relative of C. arietinum was C. reticulatum, followed by C. echinospermum and other species, while C. cuneatum was the farthest relative. In general, C. cuneatum was also genetically the farthest removed from any other species. The suggestion that C. reticulatum is the wild progenitor of the cultivated chickpea was therefore further supported.     

小麦种子贮藏蛋白质研究进展

小麦醇溶蛋白组成可以作为小麦品种鉴定的指纹图谱,其分离方法有酸性电泳、反相高压液相色谱（RP－HPLC）和毛细管电泳（CE）等手段,3种方法相互补充,而CE分辨率最高。对醇溶蛋白酸性电泳条件的改良和完善仍在进行中,利用最新的分离技术对小麦醇溶蛋白基因进行染色体定位和遗传行为分析是近年来醇溶蛋白研究的另一领域。小麦高分子量麦谷蛋白亚基（HMW－GS）与小麦面包烘烤质量密切相关,关于它的研究目前主要集中在3个方面;对各个迁3移率较近的亚基进行快速,准确分离方法的研究,HMW－GS与小麦面包烘烤质量关系的研究和通过基因工程来改良小麦的品质、提高面粉的加工特性等。低分子量麦保蛋白（LMW－Glutenin)影响小麦面粉的特性,截止目前已经获得了17个该基因的克隆,并对其基因结构进行了描述,有些低分子量麦谷蛋白亚基（LMW－GS）加入碱性面粉后改变了面筋的性质,报道了小麦醇溶蛋白,高分子量麦谷蛋白亚（HMW－GS）、低分子量麦谷蛋白亚基（LMW－GS）3个方面的最新研究进展。     

Accumulation of seed storage proteins and the taxonomy ofPoaceae

The accumulation of specific seed proteins is a taxonomically valuable feature and can be used to additionally characterize plant taxa. To date, mainly crop proteins have been analysed in thePoaceae. In this investigation seed proteins from 147 species were screened with emphasis on legumin-like proteins and prolamins. The groups resulting from evaluation of the protein profiles correspond with well-known subfamilies and tribes.Panicoideae are clearly separated fromPooideae. WithinPooideae, theBromeae plusTriticeae tribes revealed obvious similarities.Lolium, Festuca andVulpia, generally included in the tribeFestuceae, revealed a protein profile similar to the profile of theBromeae/Triticeae. Legumin-like proteins are accumulated abundantly inBambusoideae andPooideae exceptBromeae/Triticeae, however, only the species included in theAveninae subtribe produce soluble (globulin-type) legumins as already known fromAvena sativa. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Electrophoretic characterization of rice varieties using single seed (salt soluble) proteins

Summary Variation of salt soluble protein fractions of seeds has been observed in a number of rice varieties as recorded in their electrophoregram tracings: both qualitative and quantitative differences were present. Analysis of variance has been found to be useful in estimating the quantitative differences. These tracings or patterns appear to be unique for each of the varieties investigated and seem to be genetical in nature as they remain constant under different environmental conditions, and therefore could be conveniently used for variety identification.     

Electrophoretic seed albumin patterns and species relationships inVicia sect.Faba (Fabaceae)

Electrophoretic analysis of seed albumins (PAGE) covered 173 accessions representing nine species ofVicia sect.Faba. The number of albumin bands recorded in particular species varied from three inV. eristaloides to 23 inV. faba; in total, 38 bands were distinguished in the investigated material. The examined species, exceptV. eristalioides, showed intraspecific variation with respect to the number and relative staining intensity of albumin bands; individual variation was especially marked inV. faba and inV. narbonensis. Hierarchical clustering of the investigated taxa was based onBhattacharyya distances calculated from the electrophoretic data. The taxa grouped in three main clusters.Vicia faba and the rather remotely relatedV. kalakhensis formed one cluster. The second cluster was composed ofV. narbonensis distantly related toV. hyaeniscyamus. The third cluster comprised three subgroups: 1.V. johannis, V. galilaea andV. serratifolia, 2.V. eristalioides, and 3.V. bithynica. The obtained results are discussed with reference to taxonomic relationships inVicia sect.Faba.     

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affected in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent components of the photoreceptor complex responsible for phototropism in Phycomyces.This work was supported in part by an equipment grant to JAP from the Syracuse University Senate Research Committee, research grants to EDL from the National Science Foundation (PCM-8003915 and DMB-8316458), and a fellowship to EDL from the Alfred P. Sloan Foundation.     

Electrophoretic analysis of the membrane proteins of spinach chloroplasts

The polypeptide heterogeneity of the chloroplast envelope has been compared with these of other fractions of Spinach chloroplasts: thylakoïds and stroma by means of SDS-polyacrylamide gel electrophoresis. The envelope electrophoretic pattern shows many polypeptides whose mobility differ from those of the thylakoïds. The possible identity of the small subunit of RudPcarboxylase with a polypeptide of the chloroplast external membrane is discussed.     

Proteomic analysis of peanut seed storage proteins and genetic variation in a potential peanut allergen

Peanut allergy is one of the most severe food allergies. One effort to alleviate this problem is to identify peanut germplasm with lower levels of allergens which could be used in conventional breeding to produce a less allergenic peanut cultivar. In this study, we identified one peanut line, GT-C9, lacking several seed proteins, which were identified as Ara h 3 isoforms by peptide sequencing and named iso-Ara h 3. Total seed proteins were analyzed by one-dimensional (SDS-PAGE) and two-dimensional gel electrophoreses (2-D PAGE). The total protein extracts were also tested for levels of protein-bound end products or adducts such as advanced glycation end products (AGE) and N-(carboxymethyl) lysine (CML), and IgE binding. Peanut genotypes of GT-C9 and GT-C20 exhibited significantly lower levels of AGE adducts and of IgE binding. This potential peanut allergen iso-Ara h 3 was confirmed by peptide sequences and Western blot analysis using specific anti-Ara h 1, Ara h 2, and Ara h 3 antibodies. A full-length sequence of iso-ara h 3 (GenBank number DQ855115) was obtained. The deduced amino acid sequence iso-Ara h 3 (ABI17154) has the first three of four IgE-binding epitopes of Ara h 3. Anti-Ara h 3 antibodies reacted with two groups of protein peptides, one with strong reactions and another with weak reactions. These peptide spots with weak reaction on 2-D PAGE to anti-Ara h 3 antibodies are subunits or isoallergens of this potential peanut allergen iso-Ara h 3. A recent study suggested that Ara h 3 basic subunits may be more significant allergenicity than the acidic subunits.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Electrophoretic analysis of proteins fromPhycomyces mutants with abnormal tropisms

Certain phototropism mutants ofPhycomyces blakesleeanus show defective bending responses (tropisms) to stimuli besides light, such as gravity, wind, and barriers. These so-called stiff mutants are affected in four genes (madD tomadG). Using two-dimensional gel electrophoresis, we have analyzed polypeptides from microsomal and soluble fractions obtained from the wild type, four single mutants, and six double mutants affected in all pairwise combinations of the four genes. Consistent differences in spot patterns formadE andmadF mutants were found in microsomal fractions but not in soluble fractions. InmadE mutants, two spots designated E1 (52 kDa,pI 6.65) and E2 (50 kDa,pI 6.65) were altered. E1 appeared denser in the wild type than in themadE mutants, while the reverse was true for E2. The spots E1 and E2 are probably under regulatory control bymadE, perhaps involving posttranslational modification. A protein spot, F1 (53 kDa,pI 6.1), was present on the wild-type gels but absent from all gels formadF mutants. The F1 polypeptide probably represents themadF gene product.This work was supported by research grants to E.D.L. from the National Science Foundation (DMB-8316458 and DMB-8704602) and an equipment grant to C.H.T. from the Syracuse University Senate Research Committee.