Renate Manteuffel

Studies on Seed Globulins from Legumes: I. Separation and Purification of Legumin and Vicilin from Vicia faba L. by Zone Precipitation

Summary Zone precipitation has been demonstrated to be a convenient method for separation and purification of vicilin and legumin from Vicia faba seeds. Separation was achieved on columns containing Sephadex G-75 with M c I lvaine buffer pH 4.8 followed by buffer pH 7.0. After rechromatography and DEAE cellulose chromatography vicilin was isolated with a high degree of purity and free from legumin, as indicated by disc electrophoresis at pH 4.5. Vicilin was associated with two faint, less mobile bands which were interpreted as aggregates, impurities or a consequence of polymorphism. The immunological double diffusion test confirmed that vicilin was not uniform, although completely free from legumin. No cross reaction between legumin and vicilin was detected by g-el diffusion and immunoelectrophoretic investigations. Additional evidence for the nonrelation of legumin and vicilin was the production of monospecific antisera against legumin and vicilin. The results support the hypothesis that legumin and vicilin have no immunological determinants in common but are completely different chemical entities.

Studies on Seed Globulins from Legumes: V. Immunoelectrophoretic Control of Vicilin Purification by Gel Filtration

Summary Vicilin obtained from seed globulins of Vicia faba by zone precipitation was further purified by two successive gel filtration steps on Sephadex G-200 and Sephadex G-200 “superfine”. Two dimensional immunoelectrophoresis against globulin antisera revealed that crude vicilin was a mixture of serologically different proteins, contaminated with small amounts of legumin. Some proteins, occupying identical positions on Polyacrylamide gels, were clearly separated and identified by two dimensional immunoelectrophoresis. Pure vicilin, separated from accompanying proteins by gel filtration, exhibited some serological heterogeneity. Thus, new support was added to the hypothesis, that vicilin from Vicia faba is not a uniform protein, but a complex of different, though highly similar, molecules.

Isolation and Characterization of Protein Bodies of Vicia faba Seeds

Summary Cotyledonary cells of mature Vicia faba seeds contain large amounts of protein bodies. Beside the lobed nucleus a few mitochondria and starch grains are present as well as some spherosomes around the amyloplasts and along the plasmalemma. Protein bodies were isolated in different aqueous and nonaqueous media. The yield and purity of resulting organelle fractions after the isolation procedures were estimated by quantitative serological and chemical determinations as well as by electron microscopy. The contents of protein bodies exhibit the same globulin patterns compared with total salt extracts of seed flour as revealed by polyacrylamide gelelectrophoresis and two-dimensional immunoelectrophoresis. Quantitation of phytohemagglutinin by serological techniques in protein body extracts gave amounts of about 1%. The Vicia faba lectin was absent in other organs and starch grains but present in cotyledons. E dax analysis of elements in connections with scanning electron microscopy revealed the presence of phosphate, potassium, sulphur, and calcium within the protein bodies.

Studies on Seed Globulins from Legumes VI. Association of Vicilin from Vicia faba L.

Summary Samples of vicilin after dialysis, freeze drying and storage occasionally exert a series of bands on Polyacrylamide gel electropherograms which are explained as association products. Molecular weight estimations by gel chromatography and Polyacrylamide gel electrophoresis suggest the existence of monomeric, dimeric, and trimeric vicilin molecules, that are formed in solutions of low ionic strength. During Immunoelectrophoresis in agarose gels at p H 8.8 the associated vicilin reveals an increased anodic mobility, thus perhaps explaining precipitation arc asymmetry as a consequence of overlapping between vicilin types of different degree of association.

Isolation and Partial Characterization of the Protein Body Membrane from Mature and Germinating Seeds of Vicia faba L.

Summary Electron microscopical studies of Vicia faba seeds have been shown that the protein body envelope is a protein-lipid bilayer membrane with a mean diameter of about 100 A. Protein body membranes from Vicia faba seeds were separated from protein body contents by successive salt extractions after sonication of the isolated protein bodies. Purity of the remaining membrane fraction was checked by serological techniques. A protein content of about 20% was determined on a dry weight basis. Polyacrylamide gel electrophoresis in the presence of SDS revealed about 30 polypeptides with apparent molecular weights up to about 85,000 D. Among them were 15 polypeptides which showed a positive reaction after Schiff’s staining. After urea treatments of the membrane fraction differences in the gel electropherograms of urea extractable polypeptides compared with the urea treated material were observed. SDS-polyacrylamide gel electrophoresis of protein body membranes from germinating seeds revealed no detectable changes with respect to main polypeptides of the membrane fraction during the first 6 days of seed germination. Furthermore the presence of a membrane boundary was demonstrated by electron microscopical studies of protein bodies in the cotyledons from germinating seeds even after partial loss of contrasted material from the protein body contents.

Beziehungen zwischen Pflanzen und epiphytischen Bakterien hinsichtlich ihres Auxinstoffwechsels: XV. Bakterielle Besiedlungsdichte und Bakterienausbreitung auf Pflanzenoberflächen unter Laboratoriumsbedingungen

Summary Under laboratory conditions, the number of bacteria cells per plant increases rapidly, even when external infection is excluded. Surfaces of fast growing tissues are settled by bacteria just as dense as surfaces of slow growing ones. When restricted spots of roots, seeds or coleoptiles are infected, the bacteria cells spread mainly downwards but also upwards all over the plant.

Beziehungen zwischen Pflanzen und epiphytischen Bakterien hinsichtlich ihres Auxinstoffwechsels: XI. Der Einfluß von IES-bildenden, IES-abbauenden und IES-neutralen Bakterien auf den Auxingehalt der Maispflanze im Vergleich zur Wirkung von Zucker-, Aminosäure- oder Vitaminapplikationen

Summary This investigation was undertaken to decide whether epiphytic bacteria increase the auxin content of their host plant only by giving off IAA, or also by supplying other metabolites, which could affect the auxin content directly or indirectly. 29 different but not identified bacteria strains were isolated from shoots, roots, and from the hydroculture medium of corn plants. The production of IAA from tryptophan and the degradation of IAA by these epiphytic bacteria of corn plants has been investigated. 5 strains were able to produce IAA from tryptophan without being able to destroy it, 6 strains could destroy IAA but not produce it, 13 strains had both properties, and 5 strains behaved indifferent to IAA. More extractable and diffusible auxin was received from sterilized corn plants which were artificially reinfected with epihphytic IAA-producing bacteria strains than from sterile corn plants. Reinfection with strains unable to produce IAA was ineffective, this comes true in the cases of both IAA-indifferent strains and of IAA-destroying ones. This result underlines the causal connection between bacterial IAA production and auxin increase in the host plant. Simultaneous reinfection of the sterile plant material with both an IAA-producing and an IAA-destroying strain did not affect the auxin content. An application of tryptophan increased the auxin content of corn plants which were artificially reinfected with IAA-producing bacteria strains, whereas it was ineffective in the case of plants having been reinfected with IAA-destroying or IAA-indifferent strains. This result, too, corroborates the significance of bacterial IAA production for the bacteria-mediated increase of the auxin level. Application of glucose, glutamic acid, and tyrosine did not result in any influence on the auxin content of sterile, nonsterile or reinfected plants, neither by being converted into a metabolite influencing auxin metabolism, nor by stimulating bacterial growth. Obviously another factor, possibly tryptophan, limits the bacterial effect on the auxin content of the plant. Application of thiamine, riboflavine, and pyridoxal-5’-phsophate did not affect the auxin content of sterile plants. Therefore these potential bacterial metabolites are without significance for the bacteria-induced auxin increase. Application of niacide, on the other hand, considerably increased the auxin content of sterile plants. But the results of another investigation (M anteuffel , S iegl 1973) prove niacide not to be a bacterial metabolite influencing the auxin content under our experimental conditions. The results of the present investigation confirm the supposition that the IAA production by epiphytic bacteria is physiologically significant in enhancing the auxin level in the host plant.

Beziehungen zwischen Pflanzen und epiphytischen Bakterien hinsichtlich ihres Auxinstoffwechsels: XIII. Bakterielle Auxinsynthese in Pflanzenexsudaten

Summary Previous own results demonstrated a connection between IAA production by epiphytic bacteria and the auxin level in the host plant, suggesting the following circuit of substances: plant ⟶ plant exudate (tryptophan) ⟶ epiphytic bacteria ⟶ bacterial product (auxin) ⟶ plant. Now we isolate distinct steps of this circuit from their natural environment, the plant surface. Leachates from sterile coleoptiles or whole plants of maize promote the growth of the epiphytic bacteria strains S 32 and S 42, the degree of this promotion raising with increased duration of washing the plants for receiving the leachates. The bacteria utilize the tryptophan contained in the leachate, and produce an auxin which, with regard to its Rf value, seems to be IAA. The maximal velocity of bacterial auxin production is reached during the stationary phase of bacterial growth, when the main amount of tryptophans has been already consumed. In artificial culture media, representing a mixture of carbohydrates and amino acids quantitatively adopted from the known composition of the natural maize exudate, the bacteria are likewise able to produce auxin, even when thryptophan was omitted. After having been used as bacterial culture medium, maize leachates were filter-sterilized and fractionated by ether extraction. The application of the ether soluble fraction to sterile maize plants results in a clearly elevated content of diffusible auxin in the plant as detected by use of the Avena curvature test. Application of an IAA solution leads to a similar response, whereas the ether unsoluble fraction is ineffective. The bacteria-produced ether soluble material which increases the auxin content of the maize plant seems to be IAA. By washing maize plants with ether for only one minute considerable, obviously bacteria-produced auxin amounts are obtainable from nonsterile but not from sterile plant surfaces, especially after treating the surfaces with tryptophan. Our results underline the supposition the auxin production by epiphytic bacteria is physiologically significant in increasing the auxin level in nonsterile plants under natural conditions.

Beziehungen zwischen Pflanzen und epiphytischen Bakterien hinsichtlich ihres Auxinstoffwechsels: XII. Die Ausscheidung von Aminosäuren und Vitaminen durch epiphytische Bakterien

Summary This investigation was undertaken in order to detect substances which are, besides IAA excreted by epiphytic bacteria. Two possible ways by which such substances could affect the auxin content of the host plants of the bacteria are regarded: Firstly they could serve as precursors for the IAA production performed by other epiphytic bacteria, secondly they could enter the host plants and interfere with their auxin metabolism. Tryptophan would be an example of the first mode of action, niacin and niacide examples of the second one. Epiphytic bacteria, isolated from pea and corn plants and grown in batch culture, excreted into the culture medium sixteen ninhydrine positive substances, probably amino acids. Mainly living bacteria cells seemed to be able to excrete these substances. Six of nine bacteria strains isolated from corn plants excreted tryptophan, even in the absence of an organic nitrogen source. The rate of tryptophan excretion increased with increasing duration of culturing, the main amount being accumulated during the stationary phase of bacteria growth. Tryptophan was not detectable in a suspension of dead bacteria cells having been killed by autoclaving. The tryptophan produced by living bacteria cells probably contributes to the tryptophan pool existing on the plant surface. Even when cultivated in media without organic nitrogen sources, epiphytic bacteria strains produced niacin. Addition of glutamic acid or tryptophan stimulated niacin production and lead, in one of three strains tested, to niacide production, too. As tryptophan was not more effective than glutamic acid, the former seemed not to be a niacin precursor in these bacteria. There was no correlation between the abilities of the tested epiphytic bacteria strains to produce niacin and niacide and to increase the auxin content of their host plants. Moreover, the produced niacin and niacide amounts were too low for being able to exert an influence on the IAA production in the host plants as demonstrated by the comparison with previous results regarding the effect of niacide applications on the auxin content in the plant. There are also some other experimental reasons for the statement that epiphytic bacteria are able to raise the auxin level of their host plant not by excretion of niacin or niacide, but only by IAA excretion.

Beziehungen zwischen Pflanzen und epiphytischen Bakterien hinsichtlich ihres Auxinstoffwechsels: XIV. Der Einfluß der epiphytischen Bakterien auf das Wachstum, den Geotropismus und die Adventivwurzelbildung der Pflanzen

Summary Resulting from the facts that sterilized, bacteria-free plants possess less extractable and diffusible auxin than nonsterile plants which are settled with epiphytic bacteria, and that the reinfection of sterilized plants with IAA-producing epiphytic bacteria restores the auxin content, the influence of epiphytic bacteria on some auxin dependent physiological reactions was investigated. In experiments with etiolated corn and cucumber seedlings and with green corn plants, coleoptile, mesocotyl and epicotyl length, fresh, dry and ash weight of the shoot parts of sterile plants were lower than the same parameters of nonsterile plants. In plants reinfected with IAA-producing epiphytic bacteria, all these parameters were higher than in sterile plants. Pea seedlings obviously were not affected by epiphytic bacteria. Opposite to the relations in shoot parts, length, fresh und dry weight of roots were lowered by IAA-producing epiphytic bacteria. This result underlines the assumption that the effects of the bacteria are mediated by their positive influence on the auxin level in the tissues. Isolated sections from sterile corn coleoptiles develop alower rate of elongations than sections from nonsterile coleoptiles, the same is true with sections from pea epicotyls. Sections of reinfected coleoptiles have the same rate of elongation as nonsterile sections. Treating the coleoptiles with IAA previous to section isolation has the same effect as the reinfection with IAA-producing bacteria. Experiments with antibiotics exclude the possibility that the bacteria develop their effect by producing auxin in the test medium after section isolation; the superiority of the non-sterile and the reinfected sections must result from their higher auxin content at the moment of section isolation. The bacteria-induced increase of the elongation is discussed to be the cause of all the other effects of the bacteria mentioned above. After a geotropic stimulation, sterile and nonsterile corn coleoptiles develop the same degree of curvature. But the curvature degree is raised by treating the coleoptiles with IAA or with a concentrated suspension of IAA-producing epiphytic bacteria previous to geotropic stimulation. Adventitious root formation at corn seedlings is stimulated by abscising the main root but is not influenced by the epiphytic bacteria. Obviously, under natural conditions the auxin amount produced by the epiphytic bacteria is high enough to affect the most auxin sensitive one of the investigated processes, namely growth, but is not high enough to affect the less auxin sensitive ones, namely geotropic reactivity and root formation.