Heterogeneity of catalase in maturing and germinated cotton seeds

To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000.     

A relationship between seed development, Arabinogalactan-proteins (AGPs) and the AGP mediated promotion of somatic embryogenesis

Arabinogalactan-protein (AGP) epitopes are known to display developmentally regulated patterns of expression in several plant tissues. Therefore, AGPs have been suggested to play a role in plant development. Somatic embryogenesis is regulated by AGPs as well as by EP3 endochitinases. Using four different methods we have analysed the composition of AGPs in immature carrot seeds. The results obtained show that: (1) the native electrophoretic mobility of such AGPs changes during development; (2) AGP epitopes in immature seeds are developmentally regulated; (3) enzymatically released fragments of AGPs show that the composition of these molecules changes as a function of development; and (4) the biological activity of AGPs on the formation of somatic embryos changes depending on the age of the seeds. Our results suggest that degradation of maternally derived AGPs occurs after fertilization, while cellularization of the endosperm leads to synthesis of a new set of AGPs. The presence of an endochitinase cleavage site as well as the capacity to increase somatic embryogenesis only occurred in AGPs that were isolated from seeds in which the endosperm had been cellularized. Apparently, both EP3 endochitinases and somatic embryogenesis-promoting AGPs are developmentally regulated in immature carrot seeds.     

Isoenzymatic variability in seeds of some spanish common beans (Phaseolus vulgaris L. Leguminosae): Relation to their domestication centers

Phaseolus vulgaris is an important crop species. The cultivated common bean derives from wild forms in two independent domestication centers in Mesoamerica and South America. We report a study of electrophoretic patterns of seven isoenzymatic systems in 29 samples of P. vulgaris seeds. Nineteen of them are from northern Spain, four are from Mesoamerican and six are South American forms. The isoenzymatic activity of esterases, cytochrome c oxidase, acid phosphatase, alkaline phosphatase, alcohol dehydrogenase, shikimate dehydrogenase and glutamate oxaloacetic transaminase were studied. On the basis of electrophoretic patterns of seed isoenzymes, four groups can be recognized using clustering procedures (UPGMA). Comparing this biochemical information with previous morpho-agronomic studies, the possible primary domestication centers of the cultivars are discussed.     

Wheat aging: the contribution of embryonic and non-embryonic lesions to loss of seed viability

A study has been made to identify the contribution of senescent changes occurring in embryonic and non-embryonic (endosperm and aleurone layer) tissues on germination of wheat ( Triticum durum L. cv. Appulo) seed lots of 97, 93 and 70% viability. Measurements of germination and rates of macromolecular synthesis of embryos excised from dry seeds showed that as seed viability declined from 97 to ca 93%, there was a decrease in the capacity of embryos to grow and synthesise protein, RNA and DNA. However, no significant differences were observed between embryos from 93 and 70% viability seed stocks. Changes in nutrients, fresh and dry weights of embryonic and non-embryonic tissues and electrophoretic patterns of endosperm proteins by sodium dodecyl-sulfate polyacrylamide gel electrophoresis showed a reduced mobilisation of reserve material in 70%-viable seeds. α-Amylase (EC 3.2.1.1) production was also decreased. It is suggested that alterations occurring in non-embryonic structures play a significant role in seed viability loss. This role might be mediated by failing nutrient supply, but the involvement of some inhibitor or toxic substance is also possible.     

An electrophoretic analysis of the isozymes of malate dehydrogenase in several different plants

The particulate and soluble fractions of cell-free extracts from seeds, roots, and leaves of 10 different plants were examined electrophoretically for isozymes of malate dehydrogenase. Distinct isozyme patterns were observed for each plant and even for the individual tissues of each species. There were some isozymes in several different plant extracts with equal electrophoretic mobilities, but there was no isozyme band that was common to all tissues or to all plants.     

Detection of changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Disc electrophoretic investigation of Coffea arabica and C. canephora: General protein and malate dehydrogenase of mature seeds

A preliminary disc electrophoretic investigation utilizing malate dehydrogenase and general protein banding patterns obtained from mature seeds revealed detectable differences among Coffea species, varieties and cultivars. Protein differences among seed samples from individual plants of the same variety were also implied. The analyzed protein data were presented as dendrograms and compared with published genetical and taxonomical data.     

雌雄文昌鱼同工酶的表型差异

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱。首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异 ,而在同一性别不同个体之间无差异。酯酶表型较复杂 ,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

A reinvestigation of hemoglobin alterations in ground squirrels while in various hibernation activity states

Characterization of the hemoglobins of winter-hibernating, winter-active and summer-active Arctic ground squirrels (Citellus undulatus) by citrate agar electrophoresis and isoelectric focusing (IEF), pH 5.5-8.5, showed no differences in hemoglobin electrophoretic patterns. Previous studies showing alterations in hemoglobins were most likely the result of artifacts due to the use of whole blood. The Arctic ground squirrel's hemoglobin amino terminal sequence was determined for each activity state and was identical in all cases.     

Absence of alterations in antigenic determinats inSalmonella typhimurium after mecillinam-induced morphological changes

Oval forms ofSalmonella typhimurium resulting from exposure to mecillinam demonstrated electrophoretic differences in the intracellular soluble proteins as compared to the normal organisms. No differences were found, however, in the electrophoretic patterns of cell wall protein.Salmonella rods, or oval forms stained with specific fluorescein-coupled antibodies raised in rabbits against normal or mecillinam-induced abnormal forms, showed identical patterns of fluorescence. Identical lines of precipitins in Ouchterlony system were produced between any combination of antibodies and cytosol protein from normal or mecillinam-exposedSalmonella or with cell wall extracts of either forms. These results indicated that the induced ovoid transformation produced by mecillinam is not accompanied by any demonstrable alterations in the antigenic determinants ofS. typhimurium.     

Gliadin and Glutenin Variation in Seeds of Somaclones of Triticum aestivum

Gliadin and glutenin of seeds of a pollen-derived pure line “H-33” and its 9 stable somaclones of wheat (Triticum aestivurn L.) from inflorescence callus were investigated using SDSPAGE. Among the 9 somaclones 6 were identical to the “H-33” in SDS-PAGE patterns of of gliadin and glutenin. The other 3 somaclones were identical with each other, but different from “H-33”. The varied electrophoretic bands of seed protein showed that somaclonal variation had occured at gene level during tissue culture in which the somaclones were produced.     

Protein expression during seed development in Araucaria angustifolia: transient accumulation of class IV chitinases and arabinogalactan proteins

Protein accumulation and patterns during embryogenesis in the recalcitrant seeds of the gymnosperm species Araucaria angustifolia (Bert.) O. Kuntze were studied. Soluble seed proteins, chitinases, and arabinogalactan proteins (AGPs) were analyzed by means of 2-D gel electrophoresis, mass spectrometry, isoelectric focusing, Western blot, precipitation and staining with β-glucosyl Yariv reagent (β-Glc)₃Y, and gas liquid chromatography. Despite the recalcitrant nature of the seeds, the electrophoretic patterns of A . angustifolia seed proteins showed similarities with orthodox seed types. Proteins showing chitinolytic activity were observed in all seed stages analyzed, but the expression of class IV chitinases was restricted to late stages of seed development. AGPs were prominent during stages of seed development characterized by intensive cell division and differentiation, and their decrease during seed maturation might be related to cell wall modifications during the deposition of storage compounds. Gas liquid chromatographic analyzes of AGPs did not show quantitative changes in their carbohydrate moieties during seed development. A low molecular weight protein specifically expressed in mature seeds was precipitated using (β-Glc)₃Y. Amino acid sequences obtained from MS/MS analysis revealed peptides rich in valine and acidic amino acids, but devoid in amino acids normally found in AGPs polypeptides, suggesting that these peptides are not related to classical or non-classical AGPs. Possible implications of chitinases and AGPs during seed development in A . angustifolia are discussed.     

离子束介导大豆DNA导入小麦后其蛋白质和酯酶同工酶分析

对由离子束介导大豆DNA导入豫麦52获得的第四代两个转化株系9004和9002中的可育株籽粒和矮秆不育株杂交籽粒以及受体豫麦52籽粒,进行酯酶和可溶性蛋白的聚丙烯酰胺凝胶电泳分析。结果表明,可育株籽粒可溶性蛋白电泳在谱带上与受体存在差异,矮秆不育株材料的酯酶和可溶性蛋白电泳图谱谱带差异更明显。由此推测,外源大豆DNA导入受体后某些片段有可能插入受体基因组,从而导致基因突变或受体基因表达发生改变。     

Some features of the inheritance of avenins,the alcohol soluble proteins of oat

Summary The inheritance of avenin components, the prolamins (or alcohol soluble proteins) of Avena, is studied by means of gel electrophoresis. Avenin is composed of rather similar proteins which appear as a polymorphic group from a biochemical point of view. After a first preliminary investigation it showed a surprisingly high interspecific variability. The average number of its constituents increases with the ploidy level but it still is much lower than that of wheat gliadin.The avenin electrophoretic patterns of 47 samples (F₄, F₅ or F₆ seeds) resulting from 3 hexaploid crosses are compared with the parental patterns. Four kinds of inheritance are observed. Roughly 50% of progeny profiles are identical to those of one of the parents. They are composed occasionally of partial sections of parental patterns. Complete additiveness occurs rather seldom. However, in one of the crosses a significant number of progeny samples show a band, one of the very slow moving constituents, which was not present in either of the parents.The study of avenin in F₁ seeds, arising from reciprocal crosses between two homozygous parent plants, shows a significant effect of maternal gene dose in the triploid endosperm.Because of both the variability and the relatively small number of avenin constituents, these results show that typical endosperm proteins such as oat prolamin constitute a useful tool for phylogenetic studies of the genus Avena.     

Studies on lectins. XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels

A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (alpha-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all N-acetyl-alpha-D-galactosaminyl-); Ricinus communis, seeds (beta-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all alpha-D-galactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (alpha-D-glucosyl-). Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix Pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physiochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.     

Significant differences in capillary electrophoretic patterns of follicular fluids and sera from women pre-treated for in vitro fertilization

Human ovarian follicular fluids and sera obtained from women pre-treated for in vitro fertilization (IVF) were investigated by capillary zone electrophoresis. Comparison of the matching physiological liquids showed substantial differences in the electrophoretic patterns. Significant decrease in the alpha(1)- and gamma-fractions of follicular fluids of every woman were observed, whereas other fractions of the samples did not show such alterations. Since follicular fluid is a product of both, secretion by granulosa cells and diffusion from the theca capillaries, we can assume that the forced production of follicular fluid upon hormone stimulation (with gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH) and corionic gonadotroph hormone (hCG)) may play role in the uneven presence of the proteins.     

Red cell membrane in hemolytic disease Studies on variables affecting electrophoretic analysis

Significant alterations in the spectrin: band 3 and band 4.1a : band 4.1b ratios and an occasional decrease in the peak height of band 4.2 with respect to band 4.1 were found in electrophoretic patterns of red cell membranes from patients with hereditary xerocytosis. Electrophoretic comparison of whole cell, cytoplasm and membrane polypeptides implied that atypical partitioning at hemolysis could account for some, but not all, of the alterations seen in membrane patterns of xerocytes. A decrease in band 4.2 peak height as well as a variation in the profile of band 3 were produced in controls by specific manipulations of the electrophoresis protocol. Metabolic depletion of normal cells produced the type of alterations in bands 3 and 4.1 found in xerocyte membranes, whereas Heinz body production, addition of calcium to the hemolysis buffer and incubation of membranes in detergent under conditions designed to promote proteolysis did not. The presence of a higher peak height of band 4.1b with respect to that of band 4.1a in membranes of patients with various other red cell disorders correlated with an increase in the percentage of reticulocytes in peripheral circulation. The appearance of both band 3 and 4.1 abnormalities in the patterns of control cells which had been enriched in young cells by density gradient centrifugation suggested that these alterations in hemolytic disease are related to the predominance of young cells in the population.     

Seed isozyme variation in Petrocoptis A. Braun (Caryophyllaceae)

The electrophoretic patterns of seven isozyme systems (ADH, AMY, AAT, GDH, LAP, MDH, and SOD) obtained from dormant seeds from 44 accessions belonging to 12 Petrocoptis taxa were compared in order to clarify taxonomic relationships within the genus. Overall, electrophoretic zymograms showed the presence of up 28 electromorphs, of which 26 were polymorphic among accessions. Mantel tests revealed a moderate level of correlation between the geographic distance matrix and several dissimilarity matrices based on the isozyme data (r=0.3052-0.3376). The electrophoretic profiles of seed isozymes did not match closely the analytical taxonomic framework drawn from morphology. Many electromorphs are widely distributed among Petrocoptis species, and since isozyme polymorphism is present within taxa, few species-specific markers have been found. However, a relationship between the geographic origin of the accessions and several electromorphs has been noticed. Isozyme data gave moderate support to the splitting of the genus into two groups previously defined on the basis of morphology and geographic distribution (western and eastern taxa). However, some samples belonging to P. hispanica and P. pseudoviscosa were somewhat intermediate between both groups as revealed by multivariate ordination techniques. Seed isozymes did not reveal any clear taxonomic grouping among western Petrocoptis species. In fact, no single segregate of this group is supported by the electrophoretic data.