Some treatments affecting growth substances in developing wheat ears

Cytokinin, auxin and gibberellin-like substances were bio-assayed in extracts from developing ears of wheat plants grown in various conditions. Changes in cytokinin activity along the ears may be related to the earlier flowering in the middle of the ear. Ears on the main stems of plants from which all the tillers had been removed contained less cytokinin than the main-stem ears of normal tillered plants. When grain development was stopped by preventing fertilization of the ovules the ear contained more cytokinin than normal ears. With de-tillered plants, removing flag leaves before anthesis increased cytokinin, gibberellin and auxin in the ears; later removal of flag leaves did not affect cytokinin but decreased gibberellin in the ears. Conversely, removing ears before anthesis did not affect cytokinin or auxin in the flag leaves, but their gibberellin was less than that of flag leaves on intact plants. Treatment of wheat ears with zeatin did not affect grain weight or number per ear which supports the conclusion that the growth substances in the ear may be adequate for normal grain growth.     

Cytokinin Production in Relation to the Growth of Pea-root Callus Tissue

The natural occurrence of free cytokinins was examined in relationto the growth of serially propagated pea-root callus tissuecultures. The relatively slow-growing pea-root callus was harvestedat regular intervals throughout a 12-week period and fresh weight,dry weight, cell number, and cytokinin activity were determined.At the end of the culture period the fresh weight had increasedabout 34 times, the cell number about 100-fold, and the dryweight approximately 19 times over that found on inoculation.Purified ethanol extracts from pea-root callus were tested forcytokinin activity by the soybean callus bioassay. Cytokininactivity was detected in extracts made at all stages of growthtested. There was a sharp rise in cytokinin content during theearly period of culture. The peak in cytokinin activity establishedat the beginning of the phase of growth was associated witha high frequency in mitoses. As growth proceeded there was adecline in both cytokinin content and in the mitotic index.     

Estimation of substances with the cytokinin activity in studies on the correlation between the cotyledon and its axillary bud in pea (Pisum sativum L.)

The content of substances showing the cytokinin activity was estimated on decapitated and one-cotyledon-deprived pea plants during that period when the promoting effect of the cotyledon excision had not yet been manifested. Results of the bioassay showed that after the excision an increase in the level of substances with cytokinin activity occurred only in cotylars growing in axillas of these excised cotyledons. These results coincide with earlier data about the content of gibberellins and auxins in the same object.     

Changes in growth-substance contents during growth of wheat grains

Samples of developing wheat grains were extracted at weekly intervals from ear emergence until maturity and the growth substances estimated by bioassay. Immature grains contained two cytokinins; one was similar to zeatin and another, with more cytokinin activity, had different properties. Ovules contained only small amounts of growth substances but at the end of anthesis the grains had a maximum content of cytokinin. The gibberellin content increased until 3 wk after anthesis then decreased; their auxin content increased until 4 wk after anthesis but decreased as the grains ripened and lost fresh weight. The husks contained smaller amounts of growth substances than the grains they surrounded. Exudates from young stems contained cytokinins and these may originate in the roots and move to the ears through the stems. The auxin in the grains was identified as indole-3yl-acetic acid and may be derived from the phenols present reacting with tryptophan.     

Mechanism of parasitism by Cuscuta reflexa: Distribution of cytokinins in different regions of the parasite vine

Cytokinins in the different regions of Cuscuta reflexa Roxb. vine have been extracted and assayed using the Xanthium cotyledon bioassay with N⁶-benzylaminopurine as the reference cytokinin. Maximum cytokinin level was found in the haustoria-bearing region with the gradient decreasing towards the apex. The cytokinin content of the apex was less than 1% of that in the whole haustorial region. Interestingly, the concave half of the vine contained about forty times more cytokinin in the haustorial region than the corresponding convex half. The high concentration of cytokinin or cytokinin-like substances in the haustoria-bearing site in Cuscuta might be due either to a high RNase activity in the region or translocation from the host to parasite or both.     

The possible involvement of cytokinins in the pathogenicity of Helminthosporium maydis

Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.     

Seedlings of medicinal plants treated with either a cytokinin antagonist (PI-55) or an inhibitor of cytokinin degradation (INCYDE) are protected against the negative effects of cadmium

Cytokinins are plant hormones that regulate diverse processes during plant growth and development. Targeted manipulation of their metabolism or perception has already shown benefits in agriculture. Recently, we described substances that can affect plants’ endogenous cytokinin status: a cytokinin antagonist, PI-55, and an inhibitor of cytokinin degradation, INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine). The effects of these substances on seedling growth in the medicinal plant Bulbine natalensis Baker (Asphodelaceae) and the metal tolerant Rumex crispus L. (Polygonaceae) under abiotic stress caused by cadmium (Cd) were tested. Cd is known for its negative effects on plant growth and its toxicity to humans. Treatment with either PI-55 or INCYDE had positive effects on seedling shoot and root growth and the fresh weight of treated seedlings grown in the presence of Cd. Even a single application of either compound at sub-micromolar concentrations was sufficient to reverse the inhibitory effects of Cd. Our results demonstrate that modulating cytokinin status with inhibitors of cytokinin perception and/or degradation may be useful in protecting plants against the adverse effects of high Cd levels.     

Cytokinin-like activity in sea water andFucus vesiculosus L

Cytokinin activity was demonstrated in the Baltic sea waters. This activity was always higher in the near-bottom water, taken from theFucus vesiculosuszone, than in the superficial zone. A cytokinin-like substance showing properties typical of 6-(3-methyl-2-butenylamino)purine (2iP) was also present in extracts from the thallus ofFucus. It was found that exogenously applied cytokinins, in some concentration ranges, markedly increased the number of adventitious branches formed on the cut surface of the thallus fragments. The growth responses of the investigated plant tissues to the different cytokinins varied according to season and kind of cytokinin applied. The possibility of exudation of some cytokinin like substances by the algal cells into the surrounding waters was considered.     

Changes in the level of endogenous cytokinin-like substances in Acer pseudoplatanus embryos during stratification and germination

The bioassay used to detect and quantify cytokinin activity was the Amaranthus test. Free cytokinin-like substances in embryos of Acer pseudoplatanus L. fruits increased during the first 20 d of fruit stratification at 5°C, but subsequently fell rapidly to values well below the amounts present in the embryos of freshly harvested fruits. These lower levels persisted throughout the remainder of a 60 d stratification period. Bound cytokinins fell during stratification from the highest detected levels present in freshly harvested material to values which were lower by about one third. No peaks of bound cytokinin activity were detected at any stage during stratification. In embryos from fruits stored at 17°C and unable to germinate, both free and bound cytokinins remained at a very low level throughout the 60 d period. Embryos from fruits previously stratified for 60 d showed increases in both free and bound cytokinins during the first 24 h of their incubation at 20°C in light, but after longer incubation periods up to 72 h, cytokinin concentrations decreased again to levels similar to those present at the commencement of the incubation period. Determinations conducted in 1979 and 1980 showed quantitative differences, but similar qualitative changes were observed in the two years. Most of the cytokinin activity was associated with compound(s) that co-chromatographed with zeatin and zeatin riboside.     

Changes in Endogenous Plant Hormones in Cherry Tomato Fruits during Development and Maturation

Hormonal extracts of cherry tomato fruits (Lycopersicon esculentum Mill.) cv. Small Fry at different stages of fruit development and maturation were bioassayed for their auxin, gibberellin, cytokinin and growth inhibitor activities. In general, the levels of endogenous growth promoters were much higher in the young developing fruits than in the more mature fruits. Free cytokinin levels were highest in the first two weeks of development then declined rapidly. However, cytokinin activity in the ribotide fraction, after treatment with alkaline phosphatase, decreased during thefirst three weeks of development then increased rapidly over the following four weeks. Auxin levels increased during early development to reach a maximum in three-week-old fruits after anthesis. Gibberellin levels during the first two weeks of development were much lower than those of auxins and cytokinins, but then increased to reach a peak in the fourth week after anthesis. A growth inhibiting substance with Rf similar to that of abscisic acid was found in the acidic fraction of the fruit extracts. This inhibitor increased gradually during fruit growth and development and reached a peak at the age of five weeks which coincides with the green mature stage.     

N9-substituted aromatic cytokinins with negligible side effects on root development are an emerging tool for in vitro culturing

Natural cytokinins as well as the majority of their synthetic derivatives show negative effects on root growth and development. Changes in morphology, primarily linked to the inhibition of the cell division in the meristematic zone, are manifested as thickening and shortening of the primary root and impaired lateral root branching. Rational design of cytokinin derivatives can partially overcome these drawbacks and reduce the negative effects. Using our database of cytokinin derivatives, we selected several aromatic cytokinin analogs with modifications at the N9 atom of the adenine moiety. We found that tetrahydropyranyl and tetrahydrofuranyl substitutions at the N9 atom led to enhanced acropetal transport of the modified cytokinin, and both derivatives also showed weak anticytokinin activity. Consequently, changes in the distribution of the active cytokinin pool together with gradual metabolic conversion of the modified cytokinin to its free form prevent root growth inhibition that limits cytokinin utilization in micropropagation techniques.     

The effect of stratification on the endogenous levels of gibberellins and cytokinins in seeds of douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and sugar pine (Pinus lambertiana Dougl.)

Abstract Seeds of Douglas-fir and sugar pine which had been exposed to various periods of moist chilling (stratification) were extracted and bioassayed for gibberellin and cytokinin activity. In Douglas-fir a peak of gibberellin activity increased progressively during stratification and was seven times higher after 7 weeks, but declined subsequently. No change in the level of cytokinin activity was observed during stratification. However, a slight increase (1.7-fold) occurred in stratified seeds placed at 22°C for 3 days. In sugar pine a high level of gibberellin activity, approximately 20-fold higher than in unstratified controls, was present in extracts of seeds which had been stratified for 8 weeks. After 10 weeks of stratification cytokinin levels had increased approximately 200-fold. Four cytokinin-like substances were detected in seeds. Three of the substances are similar in their chromatographic properties to zeatin glucoside, zeatin riboside and zeatin, respectively. Both gibberellin and cytokinin activity declined rapidly after reaching maximum levels. In seeds with the coats removed, germination was rapid and complete in the absence of stratification. Prior to radicle emergence in coatless seeds cytokinin activity increased to a maximum (approximately a 1-7-fold increase) within 24 h. Significant levels of gibberellin-like substances were present after 8 h. Gibberellin activity could not be detected in coatless seeds which were imbibed in AMO-1618, and germination was inhibited significantly.     

Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

Cytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineered Sinorhizobium strains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed the Agrobacterium ipt gene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although the ipt gene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineered Sinorhizobium strains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.     

Effects of Short-term Red Radiation and Choline Compounds on Cytokinin Content, Chlorophyll Accumulation and Photomorphogenesis in Wheat Seedlings

Effects of choline compounds (2-chloroethyltrimethylammonium chloride and 2-ethyltrimethylammonium chloride) as well as red radiation (R) pulse on the dynamics of cytokinin changes, growth and chlorophyll (a + b) accumulation were studied during the growth and greening of etiolated wheat seedlings (Triticum aestivum L., var. Mironovskaya-808). The seedlings were grown for 120 h in the dark and then exposed for 72 h to white light. Pre-treatment of caryopses with cholines and pre-irradiation of etiolated seedlings with R inhibited elongation of both coleoptile and first leaf; but the same factors accelerated these growth responses when seedlings were exposed to white light. Chlorophyll (Chl) accumulation and the first leaf appearance from coleoptile were accelerated by the pre-treatments as well. Far-red radiation (FR) reversed all effects of R but choline pre-treatment eliminated partly R/FR photoreversibility. Two compounds with high cytokinin activity (tested on a fresh weight basis by the bioassay with Amaranthus caudatus L.) were found in shoots and first leaves. One of them had R_f, UV absorbance spectrum and the biological activity similar to N⁶-(Δ²-isopentenyl)adenosine. Another cytokinin-like substance was not identified with the used standards. Stimulation of greening by R pulse and cholines was accompanied with accelerated accumulation of both cytokinin-like substances. We conclude that the influence of R and cholines on the concentration of substances with cytokinin activities detected in the leaves might be involved in the stimulation of Chl accumulation.     

Regulators of Cell Division in Plant Tissues VUI. The Cytokinins of the Apple Fruit

The cytokinin activities of extracts of organs developed from the apple fruit bud were compared using the carrot phloem bioassay, and the identity of the cytokinins in the apple fruitlet was investigated. The activity of apple fruitlet extracts was slightly greater than the activity of pedicel extracts, and considerably greater than the activities of extracts of other organs. Extracts of the developing seeds of fruitlets were much more active than extracts of fruitlet flesh. Apple fruitlet extracts contained three principal cytokinins. One was identified as a 6-(substituted amino)purine and was either zeatin or some very closely related compound. The two other cytokinins exhibited the chromatographic behaviour of zeatin riboside and zeatin ribotide. A cytokinin extracted from vegetative apple shoots was chromatographically indistinguishable from zeatin.     

The Gibberellins of Developing Bean Seeds

Properties of gibberellin-like substances extracted from Frenchbean seeds during their development were studied. Two zonesof gibberellin-like activity were detected on paper chromatograms. Changes in activity of one of the zones correlated with changesin growth-rate of the seed. From considerations of Rf on paperchromatograms, and differential activity on dwarf peas, dwarf-1and dwarf-5 corn, it was deduced that activity of this zonewas due to substances resembling gibberellin A₁ and gibberellinA₅. Gibberellin A₅-like activity was highest in young seedsand disappeared after cell division in the cotyledons had ceased.Gibberellin A₁-like activity rose to its highest level duringthe period of rapid cell expansion in the cotyledons, afterthe disappearance of gibberellin A₅-like activity. The second zone of gibberellin-like activity was due mainlyto a non-acidic substance, which disappeared at the time GAj-likeactivity was rising to its highest level. A non-acidic substance that stimulated lateral bud growth ofdwarf peas also was detected in the extracts. It is presumedto be a cytokinin.     

New aromatic 6-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives as potential plant growth regulators

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2′-deoxy-9-(β)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N⁶-substituted adenine-2′-deoxy-9-(β)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2′-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.     

Effect of Root-growing Temperature on Growth Substances in Xylem Exudate of Zea mays

Growth substances were measured in xylem exudate from the stemstumps of detopped maize (Zea mays L. cv. Inra 200), whose rootshad been grown in culture solution at either 8, 13, 18, 23,28, or 33 ?C, between 30 and 47 d after sowing. After extractionof the exudate and gel chromatography, bioassay was used todetect three cytokinin, four gibberellin, and four growth-inhibitorfractions. It was found that the export of total cytokinin andgibberellin was greatest at a root temperature of 28 ?C, whileinhibitor export was lowest at 33 ?C, the root temperature thatwas optimal for shoot extension growth. As the root temperaturewas lowered to 8 ?C, shoot growth became progressively morerestricted, but while there was a concomitant decline in cytokininand gibberellin export, there was an increase in the exportof growth inhibitors. It is suggested that poor maize shootgrowth during prolonged low root-temperature treatments, suchas at 8 and 13 ?C, may be related to an altered balance betweenthe growth promoters and inhibitors that are exported to theshoot from the roots.     

The role of cytokinins in relation to flower-bud blasting in Iris cv. Ideal: Cytokinin determination by an improved enzyme-linked immunosorbent assay

The effect of dark and light treatment on endogenous cytokinins in internodes and buds of Iris was determined. Plant material was purified by chromatographic methods and cytokinins were assayed by an immunoassay.An indirect competitive enzyme immunoassay for the determination of zeatin- and isopentenyl-adenine cytokinins was developed. This assay, which is not dependent on the titre of the antibodies raised against zeatin riboside and isopentenyl-adenosine appeared to be specific, highly sensitive and more reproducible compared to a direct competitive enzyme immunoassay for cytokinins.Isopentenyl-adenosine was the most abundant cytokinin found, followed by zeatin: the latter counteracts bud blast when injected into dark-treated plants. Smaller amounts of isopentenyl-adenine and zeatin riboside were found. Results are in agreement with the hypothesis that deficiency of growth substances like cytokinins plays an important role in the occurrence of flower-bud blasting.A possible role for the major endogenous cytokinin, isopentenyl-adenosine, which earlier was found not to be effective in counteracting bud blast when injected into buds of dark-treated plants, is discussed.