Effects of exogenous cytokinins on root formation in pea cuttings

Benzylaminopurine (BAP) or zeatin continuously supplied through the rooting solution to cuttings of pea ( Pisum sativum L. cv. Weibull's Marma), inhibited root formation down to a concentration of 3.10⁻⁹ M . The inhibitory effect of BAP in the concentration range 10⁻⁸–10⁻⁷ M was readily reversible if the cuttings were transferred to solutions without cytokinin after treatment for 1–4 days. A slight increase in the number of roots formed was obtained after treatment with low cytokinin concentrations for 1–2 days. Evidence from microscopic studies of primordia formation indicates that BAP inhibits differentiation of primordia at an early stage in their development. Growth of already formed primordia, or root elongation, was considerably less sensitive to the inhibitory effect of BAP. The results indirectly support the hypothesis that endogenous cytokinins prevent root formation in stems of intact plants and may be of importance for the regulation of rooting in cuttings.     

Cytokinins in the vascular saps of Ricinus communis

Roots are recognised as the major sites of cytokinin synthesis and shoots receive a continuous supply of cytokinins from the roots. Although reports are available on the xylem mobility of putative free bases and their ribosides, relatively few studies on the phloem mobility of cytokinins have been reported. The origin of phloem-mobile cytokinins is uncertain but there is evidence which implicates a recirculation from the root source. This study is the first report in which zeatin and zeatin riboside from the root pressure exudate and phloem sap of Ricinus have been identified by full-scan GC-MS and quantified by GC-MS selective-ion-monitoring. In this study, the concentration of cytokinins in root pressure exudate was similar, but lower, and in the phloem sap higher than that reported previously. The concentration of cytokinins quantified in the phloem sap confirms their transport in the sieve tubes. The relatively high concentration of zeatin riboside detected in the root pressure exudate and of zeatin detected in the phloem sap indicate a possible vascular recirculation of these hormones.     

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.     

Changes in Gene Expression from Diploid to Autotetraploid Status of Lycopersicon esculentum

The cytokinin activity of the root exudate, the leaves, and the apices of vegetative and flowering white lupin plants (Lupinus albus L.) was investigated. The level of cytokinin activity in the root exudate decreased over the 11-week experimental period. Four peaks of cytokinin activity were recorded in the root exudate of 8-week-old plants after fractionation on Sephadex LH-20. Two of these peaks co-eluted with zeatin and zeatin riboside. It is suggested that the remaining peaks represent nucleotide and glucoside cytokinins. The cytokinin levels in extracts of the mature leaves fluctuated slightly over the experimental period. Three peaks of activity co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in extracts of mature leaves of 8-week-old plants. In the apices cytokinin activity could only be detected in the inflorescences of flowering plants. It would appear that cytokinins produced by the roots accumulate in the fully expanded mature leaves, but are utilized in the rapidly growing apical region and in young expanding leaves.     

Cytokinins in Rosa hybrida in relation to bud break

To assess the role of endogenous cytokinins in growth and development of Rosa hybrida , their concentrations in bleeding sap and in roots, stem, leaves, axillary shoots and bottom breaks in three stages of development were quantified. Cytokinins were purified by means of immunoaffinity chromatography and HPLC, and identified by retention time, UV spectrum and GC-MS. The major translocation form in the xylem was zeatin riboside (ZR). In all mature tissues, cytokinins of the zeatin-type were predominant, amounting to 80–90% of the total cytokinin concentration. The stems contained high concentrations of cytokinins, probably caused by lateral movement of ZR from the xylem to adjacent stem tissue and the ability of the stem to metabolize cytokinins. In young leaves the contribution of isopentenyl adenine (iP)-type cytokinins to the total cytokinin pool was about 50%, indicating that these leaves might be capable of de novo synthesis of cytokinins. In older leaves, the concentration of an unidentified cytokinin-like compound increased to more than 50% of total cytokinins. This compound, which was also found in the roots, might be a storage form of cytokinins. In young axillary shoots, about 50% of the cytokinins are iP-compounds, suggesting either import of iP-type cytokinins via the phloem or de novo synthesis of cytokinins. In buds forming bottom breaks, ZR and zeatin riboside monophosphate (ZRMP) are the main cytokinins, indicating that these buds receive their cytokinins from the roots.     

Cytokinin Activity in Lupinus albus

The cytokinin content of the root exudate and leaves of fruiting white lupin plants (Lupinus albus L.) was investigated at 2 weekly intervals after anthesis of the lowest flower on the primary inflorescence. Up to 8 weeks after anthesis the level of cytokinins in the root exudate increased. However, at 10 weeks after anthesis insufficient sap was produced for analysis. Cytokinins co-eluting with zeatin and zeatin riboside were detected in the root exudate after fractionation on Sephadex LH-20. The cytokinin levels in the mature leaves steadily increased up to 8 weeks after anthesis and thereafter remained relatively constant. Three peaks of activity, co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in the leaf extracts. The level of glucoside cytokinins in the leaves was high at 8 and 10 weeks after anthesis. Paper chromatography of extracts of fruits collected at 2 weeks after anthesis indicated that as fruit development proceeded there was a build up of cytokinin in this region of the plant. It is suggested that, in the white lupin, the cytokinins translocated to the shoot are accumulated in the leaves and in the fruits and that it is only later when there is a considerable decrease in sap (10 weeks after anthesis) production that a decreasing supply of cytokinins leads to shoot senescence.     

Dependence of cytokinin distribution in plants on their physical and chemical properties and transpiration rate

The effect of transpiration on cytokinin accumulation and distribution in 7-day-old wheat (Triticum durum Desf.) seedlings grown on nutrient medium supplemented with zeatin or its riboside was studied. The content of cytokinins in plants and nutrient medium was measured by the immunoenzyme analysis; cytokinin distribution between root cells was assessed immunohistochemically using antibodies against zeatin derivatives. The rate of transpiration was reduced 20-fold by plant placing in humid chamber. At normal transpiration, after 6 h of plant incubation on the solution of zeatin, the level of cytokinins in plant tissues increased stronger than after incubation on the solution of zeatin riboside (by 7.3 and 3.5 times, respectively, as compared with control), although the rates of both cytokinin uptake were equal. Most portions of cytokinins were retained in the roots, which was stronger expressed in the case of free zeatin uptake. A decrease in the rate of transpiration did not affect substantially the zeatin absorption from nutrient medium and the total level of cytokinin accumulation in plants, but these indices were sharply decreased in the case of zeatin riboside. In the zone of absorption of both control roots and roots treated with cytokinins, more intense cytokinin immunostaining was observed in the cells of the central cylinder. The interrelation between cytokinin distribution between the cells and apoplast, their inactivation, and transport over the plant and their form (zeatin or zeatin riboside) used for treatment is discussed.     

Fast changes in growth rate and cytokinin content of the shoot following rapid cooling of roots of wheat seedling

Changes in the concentration of cytokinins were studied following root cooling. Simultaneously, the growth rate of the second leaf was monitored with a highly sensitive growth sensor attached to its tip. Cytokinins were separated by thin layer chromatography and immunoassayed using antibodies to zeatin riboside. The extension rate of the second leaf decreased within 15 minutes of cooling the nutrient medium from 24 °C to 4 °C. The concentration of cytokinins in shoots decreased with similar rapidity. In contrast cytokinins in roots increased slightly during the initial period of cooling before declining. The sharp decrease in cytokinin concentrations in shoots 15 minutes after cooling of roots may contribute to the abrupt inhibition of shoot growth.     

Cultivars of Hexaploid Wheat of Contrasting Stature and Chlorophyll Retention Differ in Cytokinin Content and Responsiveness

The work reported here compared cytokinin content and sensitivityin a selection of hexaploid wheat (Triticum aestivum L.) cultivarsusing the following measurements: leaf cytokinins at three timepoints during light-growth and at four 24 h intervals afterlight-grown plants were transferred to darkness; sensitivityof root growth to direct applications of isopentenyl adenosine([9R]iP); and, sensitivity of germination and subsequent rootand shoot growth to 18 h imbibition of seeds in benzyladenine(BA). Accumulation of zeatin riboside-type cultivars was greatestduring light-growth in Tibet Dwarf, a wheat with an extremedwarf phenotype, intermediate in Omar standard and dwarf cultivars,and lowest in the standard and dwarf versions of Itana. Cytokininlevels were otherwise not directly correlated to plant staturein these wheats. There were no cultivar-associated qualitativedifferences in the types of cytokinins detected in this study.During the 16 h light period, the content of zeatin riboside-typecytokinins increased up to tenfold and then declined to basallevels during dark growth. Chlorophyll retention during dark-growthwas correlated with leaf cytokinin content. Data collected ata restricted number of sampling points during dark-growth suggesteda cyclic accumulation of [9R]iP-type cytokinins and the apparentcycle in Tibet Dwarf was offset by 24 h. Tibet Dwarf showedthe greatest root growth inhibition after exposure of seedlingroots to [9R]iP or imbibition of seeds in BA. Neither of thesetreatments affected shoot growth in any of the cultivars. Wheat; Triticum aestivum ; cytokinin; zeatin riboside; benzyladenine; root inhibition     

Cytokinins as inhibitors of root growth

The elongation of roots of wheat ( Triticum aestivum L. cv. Diamant II), flax ( Linum usitatissimum L. cv. Concurrent) and cucumber ( Cucumis sativus L. cv. Favör) seedlings in the dark was strongly inhibited by various native and synthetic cytokinins (kinetin, benzyladenine, isopentenyladenine, zeatin and their corresponding 9-ribosides). An inhibition of 50% was obtained for wheat roots with 3 · 10⁻⁹ M zeatin and for flax roots with 6 · 10⁻⁹ M isopentenyladenine. The ribosides were in all cases less inhibitory. The inhibition was reversed by various types of 'antiauxins' and 'antiethylenes' (such as structural auxin analogues, uncouplers, specific inhibitors of ethylene synthesis, free radical scavengers, inhibitors of ethylene action). These substances as a rule counteract also inhibitions caused by auxins. Auxins and cytokinins stimulate ethylene production synergistically, and the similar inhibitory effects of these two types of hormone can be understood if it is assumed that their effect is at least partly mediated through ethylene. The cytokinins must be considered as possible natural inhibitors and regulators of root growth.     

Changes in the Endogenous Cytokinins of Bark and Buds ofSalix babylonica as a Result of Stem Girdling

Girdling of 1-year-old Salix babyionica L. plants resulted in an early accumulation of compounds which co-chromatographed with cytokinin glucosides in both the bark and buds below the girdle. In the bark the cytokinin glucosides were present in high levels in both girdled and non-girdled plants. In the buds of non-girdled plants. however, glucoside concentration was initially low but then increased rapidly after ringing and reached a maximum level prior to any visible signs of bud swell. With the onset of lateral shoot growth the glucoside cytokinins decreased while the cytokinins that co-chromatographed with zeatin and its derivatives increased. As the cytokinin glucosides are generally considered to be storage forms, their accumulation in the bark and buds below the girdle apparently does not reflect synthesis but rather transport towards a more competitive sink. In the case of Salix plants the lateral buds would appear to have the ability to hydrolyze these glucosylated zeatin derivatives and then to utilize them for bud development. It is suggested that in the presence of a functional root system lateral buds do not synthesize cytokinins de novo, but that they do have the metabolic capacity to convert cytokinins transported to them.     

Seasonal Changes in the Cytokinin Content of the Leaves of Salix babylonica

The young and old leaves of Salix babylonica contain at least four cell division-inducing compounds which coeluted with zeatin, zeatin riboside and their glucosylated derivatives. During the course of the growing season quantitative changes in the cytokinin content of the leaves were observed. The cytokinin glucosides increased as the leaves aged. The compounds which co-chromatographed with zeatin and zeatin riboside initially increased until early autumn and then decreased as the leaves senesced. It appears as though the cytokinins transported from the roots are metabolized in the leaves and are converted to their glucosides. Although it has been reported in the literature that Salix root exudate contains very small amounts of cytokinin in late summer and autumn, these compounds increase in the leaves for most of the growing season, suggesting that the leaves may not only obtain cytokinins from the roots but may well be an active site of cytokinin synthesis. It is, however, possible that cytokinins are also transported to the leaves via the phloem, thus accounting for their accumulation in these organs.     

High level of endogenous cytokinins in transgenic potato plantlets limits photosynthesis

Introduction of the gene for cytokinin synthesis into potato genome lead to a manifold increase in the level of cytokinins (zeatin, zeatin riboside, isbpentenyl-adenine, isopentenyladenosine) in plantlets grownin vitro.The increasing cytokinin level was associated with increasing tendency to teratoma formation, to decreasing leaf net photosynthetic rate and to increasing dark and light respiration rates and CO₂ compensation concentration. During plantlet (or teratoma) ontogeny, net photosynthetic rate increased simultaneously with the decrease in cytokinin level. High level of endogenous cytokinins was associated also with lower photochemical activities of both photosystems in isolated chloroplasts.     

Cytokinin biosynthesis in roots of corn

Removal of the quiescent center (QC) from the root apex of maize (Zea mays L., cv. Kelvedon 33) initiates a set of events which culiminate in the regeneration of an intact apex with a newly formed QC. Concomitant with the formation of a new QC is a marked reduction in extractable cytokinins in the tissue of the proximal meristem. Replacing the excised QC with a Dowex (acidic cation-exchange resin) bead affects both root growth and QC regeneration. Root growth is inhibited by plain Dowex beads and Dowex beads treated with zeatin; this inhibition is reversed if the beads have been treated with CaCl₂ (±zeatin). Dowex beads treated with zeatin delay the formation of a new QC; this effect is the same whether or not the beads also contain CaCl₂. The results of this investigation support the notions that cytokinin biosynthesis in roots is a result of activities of both the QC and the proximal meristem, and that cytokinins, at least if supplied exogenously, can play a role in root morphogenesis by delaying the regeneration of the QC.Abbreviations used throughout the text PM proximal meristem - QC quiescent center - RC root cap     

Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoë) marnierianum

When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy.     

Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid

Etiolated pea seedlings ( Pisum sativum L. cv. Weibull's Marma) were used to investigate the effects of exogenous cytokinins on root growth. Benzylaminopurine (BAP) added to the growth solution inhibited the elongation and formation of lateral roots and stimulated swelling of the root tips. Similar effects were obtained with zeatin. The effects were obtained over a wide concentration range down to 0.01 μ M . Growth responses appeared only after treatment for several hours, and the duration of treatment had an important influence on the degree of the effects. BAP caused a moderate increase in ethylene production as measured in excised 10-mm-long root tips. Lowering ethylene production by treatment with cobalt ions counteracted both the inhibition and swelling caused by BAP. Treatment with silver ions also reversed the effect to some extent, indicating that ethylene is involved in the response of the roots to BAP. To further study the involvement of the increased ethylene production in the elongation and swelling response, the effects were compared with those obtained after application of 1-aminocyclopropane-1-carboxylic acid (ACC) in relation to the ethylene produced from this compound. This comparison showed that the increase in ethylene production caused by BAP was too low to explain the response of the roots. However, ACC treatment caused a considerable lowering of the content of indole-3-acetic acid (IAA) in the root tips, whereas BAP did not; instead, BAP increased the amount of IAA per root tip. It is concluded that cytokinins influence growth processes in roots via several mechanisms. A synergistic interaction between endogenous IAA, maintained at a high level by the cytokinin treatment, and the increased ethylene levels appears to explain most of the cytokinin effects during the first day of treatment.     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

Quantification of the export of cytokinins from roots to shoots of Rosa hybrida and their degradation rate in the shoot

Cytokinins from the roots may be involved in regulating rose ( Rosa hybrida ) shoot growth and development. The objective of this study was to estimate the export of cytokinins from the roots and their degradation rate in the shoot, which were expected to be correlated with plant development. Hence, the total cytokinin content of the shoot, the concentration of zeatin riboside (ZR) in bleeding sap, and the transpiration rates in three stages of development were determined. The estimations performed are based on the assumption that the cytokinin concentration in bleeding sap is representative for the cytokinin concentration in xylem sap in situ. This was verified by comparing the ZR concentration in bleeding sap and in sap obtaíned after pressurizing the root system to a level equivalent to the leaf water potential; no significant differences could be found. The import of cytokinins could not be correlated with plant development, as it increased linearly with time. The estimated relative degradation rate of cytokinins in the shoot decreased as the plants matured. The half-life of cytokinins in the shoot was found to be approximately 1 day, indicating that cytokinins are rapidly metabolized in the shoot.     

Aging of human fibroblasts in vitro. Correlations between DNA synthetic ability and cell size.

In tobacco cell suspensions, protein synthesis and mitotic activity were inhibited by amino acid analogues: p-fluorophenylalanine (pFPA) or 5-methyltryptophan (5MT). After inhibition by pFPA, when the mitotic activity recovered in the presence of phenylalanine and casein hydrolysate, the time table of the mitotic phases was permanently altered. The inhibiting effects of 5MT were effectively reversed by tryptophan addition to the medium. Therefore 5MT was selected for reversible protein synthesis inhibition in partially synchronized cell suspensions. When cytokinin was added in a culture where protein synthesis was inhibited by 5MT, no mitosis was observed after the cells were transferred to a hormone-free medium and protein synthesis restored by tryptophan. Cytokinin must again be added in order to restore mitosis. Thus, the hormone effectiveness of cytokinins required that protein synthesis remained undisturbed. The effect of the protein synthesis inhibition by 5MT upon the metabolism of N⁶-benzyladenine was investigated: the intracellular concentration of this cytokinin was not altered, whereas the metabolic pool of its derivatives was quantitatively reduced.