The Effect of Ringing on Cytokinin Distribution in Salix baylonica

Although quantitative differences were observed in the cytokinin content of mature leaves and bark of Salix babylonica it would appear as if these tissues contained the same cytokinin complement. Ringing resulted in a decrease in the level of cytokinins in the leaves and an increase in the bark, both above and below the girdle. In the leaves the decrease was due mainly to a drop in the level of those compounds that co-chromatographed with the cytokinin glucosides. These compounds were also almost undetectable in the bark above the girdle, where callus was formed. The observed increase in the cytokinin content of the bark above the girdle was due to higher activity in those parts of the chromatograms where zeatin and zeatin riboside occurred. Ringing stimulated the growth of lateral buds below the girdle. These developing buds as well as the bark below the girdle contained very high levels of cytokinins that cochromatographed with zeatin and zeatin riboside.     

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.     

Cytokinins from terminal buds of Euphoria longana during different growth stages

The presence of endogenous cytokinins were detected in the terminal buds of longan ( Euphoria longana Lam.) after purification by ion exchange and Sephadex LH-20 chromatography, and bioassay, enzymic degradation, high-performance liquid chromatography and gas chromatography-mass spectrometry. Permethylated derivatives of two highly active cytokinin glucoside compounds from dormant buds were: 6-(4-O-β-D-glucosyl-3-methyl-but-2-enylamino) purine (zeatin-O-glucoside) and 9-β-D-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin riboside-O-glucoside). Simultaneously, four active cytokinins from buds at the stages of leaf flush and flower bud initiation were identified as 6-(4-hydroxy-3-methyl-but- trans -2-enylamino) purine (zeatin), zeatin-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(3-methyl-2-butenyl) aminopurine (isopentenyladenosine, 2iPA) and N-(3-methyl-2-butenyl) adenine (isopentenyladenine, 2iP). The total cytokinin levels were low at leaf flush, with the zeatin and zeatin riboside in the buds about 70% of the total. In the transition of the terminal bud from leaf flush to dormancy, the principal cytokinins were zeatin-O-glucoside and zeatin riboside-O-glucoside. However, significant decreases in the levels of zeatin-O-glucoside and zeatin riboside-O-glucoside and increases in those of zeatin, zeatin riboside, 2iPA and 2iP were observed at flower bud initiation. It is suggested that in longan, the cytokinins that are translocated to the shoots are accumulated in the buds at the dormant stage, and that later there is a considerable increase in free cytokinins during flower bud initiation, leading to the promotion of flower bud development.     

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.     

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.     

The biosynthesis of cytokinins in crown-gall tissue of Vinca rosea

The crown-gall tissue of Vinca rosea converts labelled adenine into cytokinins. The principal initial products appear to be ribosylzeatin phosphates; zeatin and ribosylzeatin are also produced in appreciable quantities. The efficiency of conversion of adenine into cytokinins suggests a pathway of synthesis independent of turnover of tRNA. Isopentenyl adenine or its derivatives do not appear to be intermediates in the conversion of adenine to zeatin compounds. Cytokinins in V. rosea turnover rapidly and further metabolism of zeatin derivatives seems to result in their conversion into glucosides which are the main cytokinin active compounds in the tissue.Abbreviations HPLC high performance liquid chromatography - AMP adenosine monophosphate - TLC thin-layer chromatography - GLC gas-liquid chromatography     

Changes in cytokinin activity during flower development in Cosmos sulphureus Cav

Endogenous cytokinin activity was determined in the flowers of Cosmos sulphureus Cav. from bud emergence to full bloom using the soybean callus bioassay. Cytokinin activity was low early in flower development but increased prior to full bloom. In Sephadex LH-20 column chromatography of flower extracts, the cytokinins present co-eluted with zeatin, zeatin riboside and glucoside cytokinin. While the former two predominated prior to full bloom, cytokinin glucoside activity appeared to be at a maximum at full bloom. The possible relevance of these findings is discussed in relation to flower development.     

Cytokinins in the leaves of Ginkgo biloba. II. Metabolism and transport of 8 (14C) zeatin applied to shoot explants

Irrespective of their age, leaves of Ginkgo biloba metabolised applied 8 (¹⁴C) zeatin to compounds of similar chromatographic properties. Glucosylation is apparently not a normal feature of cytokinin metabolism in immature leaves. However, the application of zeatin to these leaves did result in the formation of metabolites which co-chromatographed with glucosylated cytokinins. As far as cytokinin metabolism is concerned therefore, this application of excess zeatin allowed immature leaves to behave as mature or senescing leaves. Overall metabolism was fastest in immature leaves. From the metabolites formed it would appear as if oxidation, which resulted in the formation of a metabolite which co-eluted with N-(purin-6-yl)glycine, was also important in immature leaves. In senescing leaves glucosylation was the major form of metabolism. Extraction and re-application of the polar metabolites (formed from zeatin) to mature leaves resulted in the formation of compounds which co-chromatographed with zeatin. This suggests that these compounds could serve as precursors for zeatin or could be hydrolysed to form zeatin.Very little of the applied radioactivity was exported from the leaves irrespective of their physiological age. When the metabolites, obtained after zeatin application to mature leaves, were extracted and reapplied to the leaves, export of radioactive material was much improved. The results suggest that should cytokinins such as zeatin be translocated to mature leaves of this deciduous gymnosperm their export from the leaves would be unlikely unless first metabolised. In all probability the metabolites concerned are cytokinin glucosides.The financial support of the C.S.I.R., Pretoria, is gratefully acknowledged.     

Cytokinins in Populus×robusta: Changes during Chilling and Bud Burst

Cytokinin levels in sap and vegetative buds of Populus×robusta Schneid have been determined during chilling and bud burst. From non-detectable levels in December and January, parallel increases in cytokinin levels occur in sap and buds during February and March, both in material from the field and that held at 2°C in the dark. The maximum in the sap occurs two weeks prior to natural bud burst, and 3 weeks, prior to the maximum attained in the buds. Excised twigs, forced to bud burst, show a similar pattern. The role of roots as a possible source of cytokinins is discussed. Partition chromatography on Sephadex LH-20 indicates that at least 5 cytokinins are present in buds, two of which have similar elution volumes to zeatin and zeatin riboside-The main activity in the sap is confined to a zeatin riboside-like component.     

Involvement of auxin and cytokinins in initiation of growth of isolated pea buds

Axillary buds from the second primary scale excised from 21-day-old pea(Pisum sativum L. cv. Vladan) plants were used as a modelsystem for studying the release of buds from apical dominance. The isolatedbudswere transferred onto basal medium with or without a supplement of growthregulators and cultivated up to 24 h in short-term and up to 4weeks in long-term experiments. In both sets of experiments endogenous IAA,cytokinins and the uptake of labelled zeatin were analysed. The development ofbuds was monitored by image analysis, estimation of their weight, and byanatomical studies. Generative meristems were found in isolated axillary budsalready in 21-day-old plants at the beginning of the experimental period. Theonset of bud growth was recorded as soon as 2 h after the budexcision by image analysis. The bud growth was accompanied by a rapid transientincrease of the endogenous IAA level within the first 2 h,followedby an increase of iPA within 24 h. The uptake of the exogenouscytokinin ([³H]Z) reached its peak between the 6 and 8hafter the release from apical dominance. The cytokinin analyses of bothshort-term and long-term bud cultures revealed the increase of free cytokininsand their glucosides, indicating de novo synthesis ofcytokinins in the buds themselves.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii

Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytoklnins from vegetative, female, and male buds of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The purified cytokinins were separated by reverse phase high performance liquid chromatography and analyzed by radioimmunoassay. Confirmation of cytokinin identities was by gas chromatography-mass spectrometry. Immediately prior to bud burst, all bud types contained three major cytokinins: isopentenyladenosine, zeatin riboside, and a hexose conjugate of zeatin riboside (not zeatin riboside O-glucoside). Zeatin-type cytokinins were present in relatively high concentration in vegetative and female buds. In male buds, however, relatively high levels of isopentenyladenosine were found together with low levels of zeatin-type cytokinins.     

ENDOGENOUS CYTOKININS IN THREE GENERA OF MICROALGAE FROM THE CHLOROPHYTA1

Nine axenic microalgal (Chlorophyta) strains from three genera (Protococcus, Chlorella, and Scenedesmus) were analyzed for endogenous cytokinins. Cytokinin‐like activity was detected using the excised cucumber cotyledon bioassay. Five strains showed no cytokinin‐like activity and four strains, low cytokinin‐like activity. Ethanolic extracts of the microalgae containing a mixture of deuterium‐labeled standards were purified using a combined DEAE‐Sephadex octadecysilica column and immunoaffinity column based on wide‐range specific mon‐oclonal antibodies and analyzed by HPLC linked to a micromass single quadrupole mass spectrometer with an electrospray interface and a photodiode array detector. There were similar trends in cytokinin profiles for the nine microalgal strains investigated, although concentrations did vary. Both isopentenyladenine and isopentenyladenosine were detected in all nine strains. cis‐Zeatin and cis‐zeatin riboside occurred at higher concentrations than the trans isomers, whereas trans‐zeatin‐O‐glucoside and trans‐zeatin riboside‐O‐glucoside were dominant over the cis isomers. Dihydrozeatin and its conjugates were not detected in any significant amounts. The aromatic benzyladenine always occurred at higher concentrations than benzyladenosine. The topolins were well represented with all three isomers (ortho, meta, and para) being detected, with ortho‐topolin and ortho‐topolin riboside occurring at higher concentrations than the other isomers. However, for the O‐glucosides, the meta isomers (meta‐topolin‐O‐glucoside and meta‐topolin riboside‐O‐glucoside) occurred at higher concentrations than the other isomers. No N‐glucosides were detected (isopentenyladenine‐9‐glucoside, zeatin‐9‐glucoside, dihydrozeatin‐9‐glucoside, benzyladenine‐9‐glucoside, ortho‐topolin‐9‐glucoside, and meta‐topolin‐9‐glucoside). Generally, zeatin and topolin conjugates were the dominant forms of isoprenoid and aromatic cytokinins, respectively. There was no distinct trend in the proportions of isoprenoid to aromatic cytokinins.     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.     

The accumulation and metabolism of plant growth regulators during organogenesis in cultures of thin cell layers of Nicotiana tabacum

The accumulation and metabolism of exogenously applied and endogenously produced auxins and cytokinins were studied in relation to organogenesis in thin cell layers of Nicotiana tabacum L. It was shown that, in order to obtain maximal flower bud formation, both exogenous auxin and cytokinin needed to be present during the first 4 days of culture (to the formation of a subepidermal meristematic zone) whereas cytokinins needed to be present for at least 4 days more (until formation of organogenic centres). Explants taken from floral branches have higher endogenous indole-3-acetic acid (IAA) levels compared with explants from the basal part of the stem which form only vegetative buds. This might be related to a different IAA metabolism in these two types of explants as was shown by the different accumulation of exogenously applied IAA. Both 'floral' and 'vegetative' cells layers contained comparable amounts of zeatin riboside (ZR) as their major cytokinin. Free bases, zeatin (Z) and dihydrozeatin [(diH)Z], given exogenously, were largely metabolised to their respective ribosides. The observation that Z was less effective than (diH)Z in the induction of flower buds could be related to (diH)ZR apparently not being a substrate for cytokinin oxidase.     

Investigations into the Possibility that Potato Buds Synthesize Cytokinins

In an attempt to determine whether potato buds synthesize cytokinins,tubers and potato pieces were subjected to conditions whichboth retard and accelerate bud development. Cytokinin activitywas recorded in the tubers and sprouts under different experimentalconditions. Most of the compounds detected had chromatographicproperties resembling those of zeatin, zeatin riboside, andzeatin glucoside. It would appear as if the buds and sproutsdo not synthesize cytokinins. Initial bud growth may be dependenton the supply of cytokinins within the mother tubers, whilethe increase in the sprouts could be the result of root developmentat their basal ends.     

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.     

Endogenous Cytokinins and the Breaking of Dormancy and Apical Dominance in Potato Tubers

Cytokinins are present in all parts of potato tubers, and areequally distributed between the apical, lateral, and internodaltissue when dormant. However, the breaking of dormancy coincidedwith a rapid increase in the free base cytokinin levels in theapical buds and the tissue adjacent to it. These high levelsof cytokinin in the apical tissue were maintained while apicaldominance was displayed. Once apical dominance was overcomethe cytokinin levels in the lateral buds and the tissue adjacentto them were similar to the levels in the apical regions. Thepresent evidence suggests that cytokinin glucosides are transportedto the meristematic regions of the tubers where they are hydrolysedto their free bases. Amounts of free bases in excess of thoserequired for growth are apparently again converted to storageforms (particularly zeatin glucoside) in the meristematic regionsof the tubers and in the sprouts.