Production of auxins and gibberellin-like substances by mycorrhizal fungi,bacteria and actinomycetes isolated from soil and the mycorrhizosphere of pine (Pinus silvestris L.)

Summary Production of auxins and gibberellin-like substances by mycorrhizal fungi, bacteria and actinomycetes isolated from the mycorrhizosphere of Scots pine was studied. Chromatography and biossays were used.Most of the organisms required tryptophan for auxins production. The highest biological activity exhibited substances located at R_f 0.2–0.4.The organisms produced minute amounts of gibberellin-like substances which appeared at different R_f values. It was stated that auxins production is much more common among the root zone organisms of pine than the production of gibberellin-like substances.This research was carried out under problem MR.II.16 coordinated by the Institute of Dendrology, Polish Academy of Sciences.     

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.     

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.     

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 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.     

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 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.     

Production of plant growth substances by rhizosphere mycoflora of broad bean and cotton

The culture filtrates of the rhizosphere fungi of broad bean (Vicia faba L.) and cotton (Gossypium barbadense L.) were analysed for the presence of plant growth substances of auxin and gibberellins nature. Bioassay test and chromatographic analysis indicated that these fungi, each synthesized different auxins in their culture medium. These auxins were indole compounds. Similarly the rhizosphere fungi produced in their culture medium some gibberellins and gibberellin-like substances.     

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.     

The role of endogenous growth regulators in the differentiation processes of walnut (Juglans regia L.)

The character of endogenous growth substances was investigated in developing buds, young fruits and mature walnut leaves. The relatively high content of auxins and gibberellin-like substances was found by means of bioassays in the youngest primordia of vegetative buds. The level of auxins drops in the further course of primordia transformation into the staminate catkins. The development of leaf-buds is characterized by the accumulation of inhibitory activity as revealed by theAvena bioassay, whereas the data obtained from the lettuce bioassay indicate a pronounced stimulation. The onset of terminal bud development is also accompanied by inhibitions and it is only with pistillate flower differentiation that the temporary rise in auxin level is observed. An inhibitory activity was found in these extracts using lettuce bioassay. There is a relatively high auxin level in young fruits, mature leaves and resting buds during the mid-summer period whereas the accumulation of clearcut inhibitions is signalled by the results of lettuce bioassay. The regulatory role of growth substances in differentiation may be better understood during the second year as many leaf-abnormities appear only with the outgrowing of the bud. Abnormal catkins differ in the number of florets and stamens and some even bear pistillate flowers. Fruit development is liable to deviations in the early stages of differentiation. Abnormal fruits enable us to elucidate many structural peculiarities.     

Gibberellin-like Substances from Norway Spruce (Picea abies)

By use of methanol extraction, two different consecutive partition procedures, repeated polyvinylpyrrolidone column chromatography, silicic acid partition column chromatography and the dwarf rice, lettuce, and barley half-seed bioassays, several gibberellin-like substances were detected in elongating shoots of Picea abies (L.) Karst. No significant differences in the content of gibberellin-like substances could be detected between juvenile and mature trees. The shoot axes were found to contain fewer gibberellin-like substances than the needles.     

Effects of Daylength on Endogenous Gibberellins in Leaves of Solanum andigena

Less gibberellin-like activity was detected in extracts of potato leaves under short day than under long day conditions. Significant decreases in gibberellin levels were detected after two short day cycles. Using thin-layer chromatography coupled with several solvent systems and various bioassays, at least four zones of gibberellin-like activity, A, B, C and D were found in the potato leaf. Under long days only zones C and D were apparently present. However, preliminary purification using polyamide columns revealed that substances A and B were actually present in higher levels under long day than under short day conditions. The chloroplasts of leaves exposed to short days contained less gibberellin-like substances than those from leaves grown under permanent long day conditions.     

Growth Hormone Changes in Chrysanthemum morifolium: Effects of Environmental Factors Controlling Flowering

Simultaneous quantitative analyses have been made of the endogenouslevels of auxin- and gibberellin like substances, growth inhibitors,and auxin-oxidizing enzyme activity in the cold-requiring Chrysanthemummorifolium cv. Sunbeam subjected to different daylength, lightintensity and temperature regimes known to affect flowering.While little hormone or enzyme activity was found in extractsfrom unvernalized plants, a striking rise in auxin-oxidizingenzyme activity occurred rapidly after the end of cold treatment.Increased auxin activity was also recorded shortly after vernalization.At 28 °C both enzyme and auxin activity declined over aperiod of 3–4 weeks; at 20 °C this response was delayed.Gibberellin activity at 28 °C rose steeply about 2 weeksfrom vernalization and declined several weeks later; at 20 °Ca similar response was less marked. Low light intensity treatment,which may have increased endogenous auxin levels, or exogenousauxin application reduced gibberellin-like substance levelsand cause d devernalization.Phosphon D treatment also loweredgibberellin levels and prevented flowering. An extract fromvernalized plants containing gibberellin-like substances intensifiedthe flowering of partially vernalized test plants. Persistenceof high auxin activity in vernalized plants on long days wasassociated with failure to form normal flower buds. Stem elongationrates correlated in general with levels of endogenous auxin-and gibberellin-like substances. Significant amounts of an abscisin-likeinhibitor were found in extracts of flower buds. The mechanismof natural devernalization is discussed in relation to theseobservations.     

Effects of Daylength on Endogenous Gibberellins in Solanum andigena III. Gibberellin Production by the Leaves

Using an agar diffusion technique, it was found that leaves from potato plants growing under long days produced more gibberellin-like substances than did leaves from plants growing under short days. Short day plants irradiated with red light during the long dark period and harvested during the ensuing light period, contained levels of gibberellin-like substances approximating those found in long day grown plants. Red irradiation during the long dark period also resulted in an increase in gibberellin production in short day plants. Four zones of gibberellin-like activity (A, B, C, D) were separated by thin layer chromatography in extracts from potato leaves. Red light treatment reduced the levels of peak D and brought about a concomitant increase in the levels of peak A.     

Changes in Levels of Naturally Occurring Gibberellin-like Substances during Germination of Seed of Lycopersicon esculentum Mill.

Naturally occurring gibberellin-like substances possessing acidic,basic, and neutral properties were detected, by paper partitionchromatography, in ethanolic extracts of tomato seed and ofetiolated seedlings after 72 and 116 hours' growth. Dwarf maizemutants of the d-1 and d-5 types, ‘Meteor’ pea seedlingsand young ‘Potentate’ tomato plants were used asbioassay material. Hydrolysis of seed and seedling proteinsby ficin in phosphate buffer, pH 6.2, after removal of ethanol-solublesubstances, liberated more and different ‘bound’gibberellin-like substances. It is suggested that protein hydrolysisduring germination is an important means of liberating thesesubstances at different stages of seedling development. Acidic substances were present in all the extracts prepared,but in general two with Rfs 0.25 and 0.55 in iso-propanol: ammonia:water : : 10:1:1 v/v were differentiated on d-2 and d-5 maizerespectively. Neutral substances in dry seed extracts chromatographedin the same solvent, had Rfs of 0.05, 0.35, and 0.95 and thesewere found only in the ethanolic (‘free’) extracts.They were active on d-1 and d-2 maize and ‘Meteor’pea. Basic gibberellin-like substances with Rfs of 0.05 and0.35 were found in ‘free’ extracts of both dry seedand etiolated seedlings after 116 hours' growth which were activeon d-2 maize only. Two others with Rfs 0.45 and 0.95 were extractedfrom seedlings after 72 hours' growth and these were activeon young ‘Potentate’ tomato plants. It is suggested that certain gibberellin-like substances, capableof reversing dwarfism in test plants, may be responsible formorphogenetic or other responses not involving stem extensionin the parent species. Changes were found in the levels of gibberellin-likesubstances but there was no evidence of changes in levels ofseed inhibitors relative to seed growth substances.     

Evidence for gibberellin-like substances in phloem exudate of higher plants

Summary Samples of sieve-tube sap were obtained as honeydew from aphids feeding on three species of higher plants. The honeydew was extracted, chromatographed and tested in several bioassays for the presence of gibberellin-like substances. The bioassay results indicated that gibberellin-like substances were translocated in the phloem of dandelion (Taraxacum officinale), broad bean (Vicia faba) and willow (Salix viminalis). Results obtained with willow showed that the concentration of gibberellin-like substances in the sieve-tube sap is daylength dependent, high levels being present in plants maintained under long days and low levels in short day plants.     

Changes in endogenous gibberellins in plant organs producing and utilizing photosynthates

The distribution of endogenous gibberellin-like substances in individual organs ofZea mays L., cv. CE 250, plants was investigated during the transition from the vegetative to the generative phase of development. The gradient of the content of gibberellin-like substances and photosynthetic activity in leaf segments was followed in different parts of the Jeaves, as well as the changes in the content of gibberellin-like substances in leaf segments during an exposure in the light and in the dark. The gradient of the content of endogenous gibberellin-liko substances in the leaves, in the stem and in the spike is interpreted in terms of possible relationship of these compounds to the regulation of sink — source.     

Studies on two Gibberellin-like Substances in Young Shoots of Tomato (Lycopersicon esculentum Mill.)

Two gibberellin-like substances were found in the acidic fractionof shoot extracts of the tomato (Lycopersicon esculentum Mill.,cultivar Potentate). These were resolved by paper chromotographywith iso-propanol/ammonia/water (10:1:1) as the developing solventbut not with n-butanol/1.5 N ammonia (3:1). Both substanceswere active in the dwarf maize bioassay on mutants d-1, d-2,d-3, and d-5, and appeared to be more active on d-5 than d-1.Neither was active in the Meteor Pea assay. Neutral and basicfractions were inactive. The relative amounts of these two substances varied accordingto the age of the tissues from which they were extracted andthis feature is discussed in relation to future studies on thephysiology of gibberellin-like substances in vivo.     

Gibberellin-like Substances in the Sporophores of Agaricus bisporus (Lange) Imbach

Sporophores of cultivated Agaricus bisporus (Lange) Imbach,were shown to contain a gibberellin-like substance active inthe dwarf maize (d-5), -amylase and other bioassays. Ethyl-acetateextraction followed by paper, column, and thin-layer chromatographyrevealed the presence of one major active substance. Ficin hydrolysisof dried sporophore powder, after the complete removal of freesubstances, released more gibberellin-like substances, one ofwhich appeared identical to the free compound. The free substance was predominantly in the lamellae and residualpileus tissue. The major active substance released by ficinoccurred mostly in the lamellae but also in substantial equalamounts in both stipes and pilei. No activity was found in extractsof dikaryotic vegetative mycelium on malt agar. The level ofactivity in extracts from sporophores stored at – 20 °Cfell sharply after 7 d, and then remained constant over a periodof 6 weeks. The content of gibberellin-like substances in youngand old whole sporophores showed wide variation between experiments.In most cases young 2-d tissue had higher levels than old, 11-dtissue on a fresh-weight basis. Purified sporophore extractsand authentic gibberellins had no stimulating effect on growthof sporophores or of cultured vegetative mycelium. The inhibitorsof diterpene biosynthesis, CCC, and AMO-1618 induced a smallincrease in mycelial growth rate. Ethyl-acetate extraction ofhorse-straw compost prior to inoculation with Agaricus bisporusshowed the presence of gibberellin-like activity in significantamounts.     

An Agar-diffusion Technique Applied to the Study of Gibberellins in Tomato (Lycopersicon esculentumMill.) Plants

Chromatography of extracts of agar blocks on which shoot tipsof tomato had stood for 24 h revealed the presence of at leasttwo gibberellin-like substances. Gibberellin-like activity detectedby the dwarf pea bioassay was shown to be associated with activityin promoting elongation growth in tomato seedlings. The activesubstances were acidic; tests for the presence of ‘neutral’,‘basic’ and ‘bound’ gibberellins inextracts of the agar blocks gave negative results. Amounts ofgibberellin-like substances obtained from the shoot tips wereincreased by the incorporation of EDTA into the agar blocksbut were apparently unaffected by the temperature (within therange 5–25 °C) at which the diffusion was carriedout. It is suggested that the technique measured levels of diffusiblegibberellins in the tissues at the time of their excision ratherthan rates of gibberellin synthesis during the diffusion period.