The effect of carbon and nitrogen sources on auxins and gibberellin-like substances synthesis by bacteria isolated from the roots of pine seedlings (Pinus silvestris L)

It was found that in the presence of sodium succinate synthesis of auxins and gibberellin-like substances by microflora in the rhizosphere of pine seedlings was higher than in the presence of the other carbon sources. The smallest amounts of these substances were found in media with sodium pyruvate. The highest C-content (C:N = 60:1) was not optimal for the synthesis of plant growth regulators. Ammonia ions inhibited the production of gibberellin-like substances. The effect of different nitrogen sources and C:N ratio on the synthesis of auxins was different depending on bacterial strain used.     

The effect of zinc on the biosynthesis of tryptophan,andol auxins and gibberellins in barley

The action of zinc on the growth of barley and the biosynthesis of indol compounds and gibberellin-like substances was investigated in a number of concentrations of zinc from doses stimulating growth to toxic doses. The seeds were soaked before sowing in solutions of zinc sulphate (5.10^?5 to 5.10^?1% Zn), and the plants cultivated for 7 days in water. Lower concentrations of zinc increased both plant growth and the biosynthesis of tryptophan and auxins. At the optimum concentration of 5.10^?3% Zn this increase in tryptophan amounted to 241% of the variant without zinc; in substances with an R_F corresponding to indolyacetic acid, the increase determined by the biological test, was 207% as against the variant without zinc. Higher concentrations of zinc inhibited growth, the tryptophan content was decreased to below that of the control without zinc and the auxin content also fell to below the control values. Zinc also influenced the content of gibberellin-like substances in the plants. At a concentration of 5.10^?3% Zn the increase in the growth activity in the gibberellic acid area of the chromatogram was 294% of the variant without zinc. At toxic concentrations of zinc, the content of gibberellin-like substances fell to below that of the controls. The finding that zinc acts simultaneously on the biosynthesis of auxins and gibberellins is also evidence for the common action of growth substances of various chemical types on plant growth.     

Production of Plant Growth-Regulating Substances by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae

Glomus mosseae, a representative species of Endogonaceae (Phycomycetes) able to form vesicular-arbuscular mycorrhiza, was investigated for phytohormone production. Spores of G. mosseae were axenically germinated in water, and the resultant mycelial growth was assayed by standard procedures for extracting plant hormones from microbial cultures. Paper partition chromatography and specific bioassays were used to separate and identify plant growth-regulating substances. The microorganism synthesized at least two gibberellin-like substances, one with R_f corresponding in position to authentic gibberellic acid, and four substances with the properties of cytokinins.     

Endogenous Growth Substances in Normal and Dwarf Mutants of Cortland and Golden Delicious Apple Shoots

Endogenous growth substances in the shoots of dwarf mutants of Cortland (Cortland 65–6 and Cortland 65–7) and Golden Delicious (Golden Auvil Spur and Yellow Spur) were investigated in relation to dwarfism. The shoots were extracted at three stages of growth, namely: 1) rapid elongation growth, 2) formation of terminal bud, and 3) cessation of cambial growth. The growth substances were partially separated by thin layer chromatography and bioassayed using the Avena first internode and Lepidium seed germination tests. Shoot extracts from the normal Cortland and Golden Delicious plants contained higher levels of auxins at all stages of growth than those from dwarf mutants. Growth-promoting activity was found in two zones on chromatograms developed with isopropanol: ammonia: water (10:1:1 v/v/v):R_f 0.4 to 0.5 and R_f 0.8 to 0.9. The Lepidium seed germination test showed the presence of a highly inhibitory zone between R_f 0.3 and 0.4 in the extracts of dwarf and normal plants. However, the peak of germination inhibition activity was sharp for dwarf mutants and less for extracts of normal growing plants at 3 stages of growth. The inhibitory substance(s) was further purified on TLC plates by successive separations in different solvent systems. Two substances which inhibited the Lepidium seed germination were found, one close to the R_f value of synthetic abscisic acid and the other appeared to be similar to xanthoxin in R_f value, biological activity and UV absorption spectra.     

Role of growth regulators in differentiation processes of maize (Zea mays L.) organs

The following paper deals with the character of endogenous auxins and gibberellinlike substances in the maize tassel and ear primordia during differentiation. Using bioassay the character of substances extracted from tassel primordia, internodes below the tassel, ear primordia and stem base was determined and correlated with the course of morphogenesis and differentiation. A low level of auxins and a high content of gibberellin-like substances accompanies the differentiation of terminal tassel. The differentiation of an ear is associated with an increment in auxin content while the level of gibberellin-like substances decreases. The character of growth substances in primordia remains practically unchanged in the course of further differentiation. The inhibitions appear in the plant and probably start numerous morphological reductions in the pistillate inflorescence structure or inhibit the growth of lateral primordia on the stemetc. The treatment of plants with maleic hydrazide at the beginning of tassel differentiation shifts the normal levels of endogenous regulators and brings about the transformation of tassel primordia into an ear. This transformation is accompanied by a marked rise in gibberellin-like substances, by an increment in auxins and the appearance of inhibitors.     

Ein Beitrag zur Analyse der Gibberelline in Samen von Pisum sativum L.

Summary Unripe seeds of Pisum sativum were analyses for gibberellins and gibberellin-like substances. Forty kg of seeds were extracted with methanol, the methanol was evaporated and the residue distributed between water and ethylacetate, and between water and butanol at different pH-values. The acidic ethylacetate and the butanol fractions were separated by thin layer chromatography. In the acidic ethylacetate fraction a new substance tentatively named gibberellin A_x was isolated by preparative thin layer chromatography. In different solvent mixtures this substance migrates closely behind gibberellin A₃. It is biological active in a dwarf pea and in dwarf maize mutant bioassays. IR and fluorescense spectra point to a gibban structure. Furthermore gibberellin A₅ was found to be the main gibberellin in pea seeds. It was identified by its R_f-values and the R_f-values of its methylester in different solvent systems as well as by its characteristic relative biological activity in three biological test systems. Most probably gibberellin A₆ is also present in the pea seeds, but because the amounts detected were small, no clear evidence can be presented.In the butanol phase two gibberellin-like substances could be separated by thin layer chromatography.

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Mit Hilfe der Deutschen Forschungsgemeinschaft.     

The influence of gamma radiation on the biosynthesis of indoles and gibberellins in barley the action of zinc on the restitution of growth substance level in irradiated plants

Investigations were made on the effect of exposing barley seeds to gamma-radiation (5–40 kR), alone and in combination with the application of zinc (soaking the seeds in solutions containing 5.10^?5–5.10^?1% Zn for 12 hours before sowing) on growth and on the content of tryptophan, indole auxins and gibberellin-like substances in seven-day plants. Radiation decreased both growth and the content of tryptophan (e.g. by about 53% at 30 kR), of indole auxins (by about 60% auxin in the zone of IAA on the chromatogram at 30 kR), and also the content of gibberellin-like substances (by about 67% gibberellin content in the zone of GA₃ on the chromatogram) of plants. The irradiation of standard samples of tryptophan, indolyl-acetic acid and gibberellic acid alone with many times greater doses (up to 1000 kR) did not lead to marked radiochemical degradation of these substances. It can be assumed that radiation damages the enzyme systems “synthesizing” natural growth substances in plants. The damaging effect of radiation on auxins is already displayed in the synthesis of tryptophan, which is inhibited. Zinc interacts with the damaging effect of radiation on growth. Optimum concentrations of zinc (5.10^?3% Zn) counteract the effect of radiation, up to doses of about 12 kR, on the growth in height in 7-day plants so that it is equal to the controls. Normal content of tryptophan and auxin in the position of indolecetic acid on chromatograms can only be reached by the addition of zinc when the dose of radiation was not greater than about 8 kR, which is less than the influence exerted by zinc on the restitution of growth. On the other hand, the biosynthesis of gibberellin-like substances at the position of gibberellic acid on chromatograms can be restored by zinc to their original level to doses of up to 30 kR. The increased biosynthesis of auxins and gibberellins caused by zinc in irradiated plants is explained by the activation of the remaining and non—damaged enzyme systems carrying out this biosynthesis. The activation of the biosynthesis of growth substances by zinc will also contribute to the restitution effect of zinc on the growth of plants from irradiated seeds.     

Gibberellin-like Activity in Berries of Seeded and Seedless Tokay Grapes

Gibberellin-like activity in berries of both `Tokay' (seeded) and `Seedless Tokay' grapes (Vitis vinifera L.) at different stages of development was determined. Activity in both seeded and seedless berries was mainly in the acidic ethyl acetate fraction. In seeded berries there was very high activity beginning at the early fruit-set stage and persisting for about 3 weeks after which the activity fell to low level and vanished by the middle of July. On August 1 there was another activity peak. The activity in `Seedless Tokay' was similar except the decline was considerably more rapid and a secondary peak was reached in mid-June instead of August. The gibberellin-like substances in the acidic ethyl acetate fraction occurred mainly between R_F 0.3 and 0.6 when the chromatogram was developed with ammoniacal isopropanol.

The gibberellin-like substances from both types of berries were active in dwarf pea, dwarf corn, and lettuce hypocotyl bioassays, but not in the cucumber hypocotyl test. The role of gibberellin-like substances in berry development is discussed.

     

Regulative Systems in the Developing Citrus Fruit I. The Hormonal Balance in Orange Fruit Tissues

The regulative systems of auxins, gibberellin-like substances and their inhibitors in citrus fruit were studied. Masking caused by a high content of inhibitors was eliminated by using a refined method of solvent partition. Considerable amounts of auxins and gibberellin-like substances were detected in all stages of fruit development. The auxin system of the citrus fruit is highly complex and consists of various elements which undergo dynamic changes throughout the growth period. The identity of the auxins was studied using IAA-2-¹⁴C, and 88% of the radioactivity specifically migrated into the etheric pH 6.0 fraction. Although the prominent zones of promotion do not coincide with IAA, it can be concluded that the auxin promoters are probably not the “citrus auxin”. Abscisic acid-like inhibitors were found to accumulate in the external layers of the fruit, increasing in content as time advances.     

The effect of culture medium composition and incubation time on synthesis of gibberellin-like substances by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.)

It was found that synthesis of gibberellin-like substances by ten strains of Coryneform bacteria isolated from the roots of pine seedlings depended on both the composition of the medium and incubation time. More of these substances were produced in mineral medium with glucose in complex medium with casamino acids and yeast extract. Most gibberellin-like substances were found in 7 or 14-day old cultures. Culture supernatant fluids of most of the bacteria tested contained several gibberellin-like substances which on chromatograms run with the solvent system benzene, acetic acid (10:3, v/v) were located at Rf 0.0-0.3; 0.4-0.6 and 0.8-1.0.     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

Production of gibberellin-like substances by fungi isolated from mycorrhizae of pine (Pinus silvestris/L.).

Gibberellin-like substances were found to be produced by fungi isolated from mycorrhizae of pine. Among the isolates studied gibberellin-like substances producers were most numerous among the Basidiomycetes forming ectotrophic mycorrhiza and among non-sporulating fungi forming no mycorrhiza. In most strains the gibberellin-like subtances were elaborated after 10--20 days of incubation. The Rf values of the gibberellin-like compounds were different in various strains, but in most of them substances showing gibberellin activity appeared on the chromatograms run with benzene, acetic acid (10:3 v/v) at Rf 0.2--0.6.     

Gibberellin-like Substances From Vegetative Tissue of a Conifer, Arizona Cypress

Vegetative shoots of Arizona cypress (Cupressus arizonica Greene) were found to contain an estimated 40 to 70 μg of gibberellin-like activity per kg. Based on elution patterns of silicic acid and celite partition columns, mobilities on thin layer chromatograms and specificity of the cucumber, d-3 dwarf maize, dwarf pea, and barley half seed bioassays it was possible to determine that the tissue contained at least 5 acidic, ethyl acetate-soluble gibberellin-like substances. The major one would appear to be GA₃. In addition, GA₉, GA₄, and/or GA₇-like compounds, and 2 unidentified gibberellin-like substances are present.     

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA(1)

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A₁ solutions for 24 hours at 20°. Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA₁, and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA₁ brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA₁. Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.     

The diffusion of gibberellins into agar and water during early germination of Pharbitis nil Choisy

Agar diffusion of imbibed seeds yielded significant amounts of diffusible Gibberellin-like substances. An analysis of the extractable and diffusible gibberellin-like substance, including an analysis of the remaining imbibition water of the seeds, indicated that a significant part of these gibberellin-like substances could be attributed to a net biosynthesis of these substances in the imbibing seeds. At the same time it was found that water diffusion yielded considerably more gibberellin-like activities than comparable agar diffusions i.e. 10 to 12 fold in general.Agar as well as water diffusion showed a temperature effect with regard to the yield of gibberellin-like substances particularly during the first 6 h of diffusion. The yield of these substances is lower at 10°C, and remains lower as shown with consecutive diffusions, in comparison with the yields at 20°C or 30°C.With both agar and water diffusion the sum of activities obtained with consecutive diffusions is always higher, often considerably higher, than equal periods of continuous diffusion which is probably due to inactivation and/or interference of inhibitory substances with the bioassay responses. Finally, water diffusates of both seeds and seedlings of the normal growing cv. Violet of Japanese morning glory contained considerably more gibberellin-like activities than those of the dwarf cv. Kidachi which indicated that normals synthesize more gibberellins than dwarfs.     

Production of auxins by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.).

Qualitative and quantitative studies were carried out on the production of auxins by Coryneform bacteria, the only bacterial types isolated from roots of pine seedlings. Almost all isolates were capable of producing auxins in tryptophan containing media. In media without this amino acid only trace or no auxins were produced. Most of the bacteria studied synthesized auxins located on the chromatograms run with isopropanol, ammonia, water (10:1:1 v/v) at Rf 0.3--0.5. Moreover substances with Rf values 0.05--0.2 and 0.8--1.0 were produced by some strains. No plant growth inhibitors detected with the Avena coleoptiles biotest were produced by the bacteria studied.     

Antioxidant Enzyme Isoforms on Gels in Two Poplar Clones Differing in Sensitivity After Exposure to Ozone

The effect of acute ozone (O₃) fumigation on isozyme patterns of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) in mature (ML) and young leaves (YL) of two poplar clones, contrasting in O₃-sensitivity was analysed. Untreated leaves of both the O₃-sensitive (O₃-S) clone Eridano of Populus deltoides×P. maximowiczii and the O₃-resistant (O₃-R) clone I-214 of P.×euramericana showed four distinct SOD isoforms with a relative mobility (R_f) of 0.54 (MnSOD), 0.60 (Cu/ZnSOD), 0.65 (unidentified), and 0.71 (Cu/ZnSOD). After O₃-fumigation the activity of the SOD isoforms showed only quantitative variations with respect to control plants. In ML of untreated O₃-R plants seven POD isoforms (R_f= 0.13, 0.19, 0.34, 0.59, 0.64, 0.70 and 0.75) were found, while in YL one isoform (R_f= 0.34) was undetected. Only three POD isoforms in both ML and YL of untreated O₃-S plants were resolved. The electrophoretic pattern of POD in O₃-S leaves was greatly modified by acute O₃-fumigation with the appearance of new isoforms in both YL and ML and the disappearance of an isoform (R_f= 0.13) in YL. Additionally, O₃-exposure induced the appearance of two APX isoforms in YL (R_f= 0.66 and 0.70), and one isoform in ML (R_f= 0.70) of the O₃-S clone. By contrast, the activity of the three APX isoformes (R_f= 0.64, 0.70 and 0.76) detected in O₃-R leaves showed only quantitative variation with respect to untreated plants. From these data it is concluded that: 1) in these poplar hybrids antioxidant enzyme activity is developmentally regulated and greatly affected by acute O₃ stress treatments and 2) the different enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes, could partly explain their distinct O₃-sensitivity.     

The phenoloxidases of the ascomycete Podospora anserina: The three forms of the major laccase activity

In Podospora anserina three laccase activities (I, II and III) were identified. Present results show the existence of an additional lacaase (an anodic protein; MW 80,000; R_f 0.07). Laccase IV derived from the dissociation at acid pH (4.5) of a protein which showed identical molecular weight (390,000) and R_f (0.1) to the oligomeric laccase I. The recovery of laccase I activity when starting from laccase IV (purified by means of isoelectric focusing) suggests that laccase I itself was the source of laccase IV. In turn, laccase IV gave rise to the laccase III after electrophoresis or dialysis at basic pH (8.5).     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Gibberellin-like activity in bleeding-sap of root systems ofHelianthus annuus detected by a new dwarf pea epicotyl assay and other methods

Summary Gibberellin-like activity was found to be present in bleeding-sap obtained from root systems ofHelianthus annuus. The presence of activity was detected with a newly described simple and sensitive dwarf pea epicotyl assay, and by the barley endosperm and dwarf maize (Zea mays) tests.The gibberellin-like substance, or substances, are acidic and ethyl acetate soluble, having anR _f value of 0.6–0.7 in paper chromatography with isopropyl alcohol: ammonia: water (1011 v/v).The quantity of gibberellin-like activity produced by the roots was estimated at approximately 0.05 g GA₃-equivalents/plant/day.Other evidence for gibberellin synthesis in roots is discussed, together with the possibility that shoot and root have significant inter-relationships with regard to the production and utilisation of other types of growth hormone.

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Zusammenfassung Am Blutungssaft des Wurzelsystems vonHelianthus annuus wurde ein gibberellinartiges Wirkungsvermögen festgestellt. Das Vorhandensein dieser Aktivität wurde mittels eines neubeschriebenen einfachen und empfindlichen Tests an Zwergerbsen-Epikotylen sowie mit dem Gerstenendosperm- und dem Zwergmais-Test entdeckt.Die gibberellinartigen Stoffe haben Säurecharakter und sind in Äthylacetat löslich. IhrR _f -Wert wurde papierchromatographisch mit Isopropanol: Ammoniak: Wasser=1011 v/v als Laufmittel zu 0,6–0,7 ermittelt.Das Maß der von der Wurzel produzierten gibberellinartigen Aktivität lag bei etwa 0,05 g GH₃-Äquivalenten pro Pflanze und Tag.Andere Hinweise auf eine Gibberellinsynthese in Wurzeln werden besprochen und ferner die Möglichkeit, daß in Sproß und Wurzel eine signifikante Wechselbeziehung hinsichtlich der Produktion und Verwendung anderer Wuchsstoffarten besteht.

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With 5 Figures in the Text