Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.     

Mass Spectroscopic Identification of Cytokinins: Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea Crown Gall

Mass spectrographic and chemical studies of the permethyl and trimethylsilyl ethers of two new cytokinins isolated from Vinca rosea crown gall callus cultures by Peterson and Miller (Plant Physiol 59: 1026-1028) indicate that they are 6-(4-0-beta-d-glucopyranosyl-3-methyl-trans-but-2-enylamino) purine (glucosyl zeatin) and 9-beta-d-ribofuranosyl-6 (4-0-beta-d-glucopyranosyl-3-methyl-trans-but-2-enylamino) purine (glucosyl ribosylzeatin). The nature of the mass spectra of the permethylated cytokinins suggests that these derivatives may have considerable utility in the detection of low levels of cytokinins in plant material.     

A cytokinin complex from the developing fruits of Aegle marmelos

Following ion-exchange, Sephadex LH-20 and paper chromatography of the methanolic extracts of young, developing fruits of Aegle marmelos Correa, cytokinin-like activity in the soybean callus assay was detected in six fractions. Of the four butanol-soluble compounds, two were tentatively identified as zeatin and zeatin riboside, and the others as zeatin glucoside and zeatin riboside glucoside. The major cytokinino of the butanol-insoluble fraction is probably zeatin nucleotide. The levels of compounds with cytokinin-like activity were high during the early phase, and low in the subsequent period of fruit growth. The activity resembling that of cytokinin glucoside increased with maturation of the fruit. The content of free cytokinins in the fruits was more than that released from the tRNA.     

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.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

Cytokinin Activity in Lupinus albus L: IV. Distribution in Seeds

Endogenous levels of cytokinin activity were examined in Lupinus albus L. seed at intervals of 2 weeks after anthesis using the soybean callus bioassay. High levels of cytokinin activity per gram seed material were present in the seeds at 2, 4, and 6 weeks after anthesis. The cytokinin activity per gram seed material was low at 8 and 10 weeks after anthesis. Cytokinin activity associated with each seed was greatest at 6 weeks after anthesis. The majority of the activity in the seeds at 4, 6, and 8 weeks after anthesis was in the endosperm. Cytokinin activity was also detected in the testas and embryos at 4, 6, 8, and 10 weeks, and the suspensors at 4 weeks. Column chromatography of extracts of the different seed fractions on Sephadex LH-20 indicated that the cytokinins present coeluted with zeatin, zeatin riboside, and the glucoside cytokinins. It is suggested that cytokinins are accumulated in the seeds and are stored in the endosperm mainly in the form of ribosides and glucosides of zeatin. The reduction in cytokinin activity in the seed coincides with the reduction in endosperm volume and embryo growth and suggests that these compounds are utilized during the course of seed maturation.     

Endogenous Cytokinins in Developing Fruits of Seeded and Seedless Citrus Cultivars

This report describes the isolation and identification of endogenouscytokinins from Citrus ovaries. Cytokinin active fractions wereobtained by extraction with 80% (v/v) ethanol, followed by purificationwith hexane, n-butanol and a polyvinylpyrrolidone and SephadexLH-20 column chromatography. Five fractions with cytokinin activity were found in the organicphase, using the tobacco callus assay. The main active compoundsin these fractions were separated by HPLC, bioassayed and identifiedby GC—MS as ribosyl zeatin, zeatin and isopentenyl adenosine.Hydrolysis of the first fraction with B-glucosidase gave cytokininactive compounds that in paper chromatography had R_F's similarto those of zeatin and ribosyl zeatin. Treating the aqueousphase with alkaline phosphatase produced a cytokinin activecompound that in paper chromatography had the same R_F as isopentenyladenosine indicating that their ribotide was probably the majorphosphorylated cytokinin present in Citrus ovaries. Key words: Citrus, cytokinin fruit set and development     

Changes in the Cytokinins of Radish Roots during Maturation

The cytokinins of developing radish roots were extracted, partially purified, and separated by thin-layer chromatography into three distinct bands of activity. One band was identified chromatographically as zeatin ribonucleotide and another was indistinguishable from a mixture of zeatin and zeatin ribonucleoside. The third band was not identified, but it was not a derivative of zeatin or of isopentenyladenine. The unidentified cytokinin had physiological properties quite different from those of the zeatin derivatives. The zeatin-based cytokinins increased in radish roots with the onset of cambial activity, and reapplication of these cytokinins to cultured primary roots stimulated cambial activity. The unidentified cytokinin became abundant only after extensive secondary thickening had occurred, and it was localized almost entirely in the xylem. It did not stimulate cambial activity in cultured roots. The evidence indicates that zeatin and its derivatives regulate cambial activity in radish, and that the unidentified cytokinin may be synthesized in the roots and transported to the shoot.     

Cytokinins from a variant strain of cultured soybean cells

A strain of soybean cells capable of growing on a tissue culture medium lacking a cytokinin produced at least 3 compounds active in the soybean cytokinin assay. The characteristics of these compounds were consistent with their being zeatin in the free form, zeatin ribonucleoside and zeatin ribonucleotide. Although the conversion from a cytokinin dependent to independent condition in this strain parallels the change of normal cells to crown gall tumor state in terms of the capacity to synthesize cell division substances, the soybean factors are distinct from the nicotinamide derivatives reported for tumor cells of Vinca.     

A Cytokinin Complex in the Developing Fruits of Lupinus albus

The apices of Lupinus albus L. were analysed for cytokinin activity at three stages of development. Little cytokinin activity could be detected in the apices at the time of flowering. However, a considerable amount of activity was detected as the fruits developed. Separate analyses of seed and pod material indicated that there was a high level of cytokinin in both these parts of the fruit. After fractionation of the peaks of activity obtained from paper chromatograms on Sephadex LH-20, four peaks of cytokinin activity were recorded. Two of these co-eluted with zeatin and zeatin riboside. A third peak at an elution volume of 360–440 ml could be hydrolysed with β-glucosidase to give activity at elution volumes corresponding to those of zeatin and zeatin riboside. This strongly suggested that both glucosylated zeatin and glucosylated zeatin riboside were present in the developing fruits of L. albus. The fourth peak at an elution volume of 160–280 ml did not disappear upon hydrolysis with β-glucosidase, and it is possible that it represented a nucleotide cytokinin.     

Changes in the hormonal status of the Taraxacum officinale Web. ovary at early stages of embryogenesis

The hormonal status of the Taraxacum officinale Web. ovary was quantitatively assayed for the first time during early stages of embryogenesis. Apparent concentrations of endogenous cytokinins were measured using two systems of enzyme-linked immunosorbent assay (ELISA). The ELISA systems differed from one another by the specificity for the main endogenous forms of zeatin. The specificity of two heterological ELISA systems based on zeatin- and kinetin-specific antisera was studied. A new immunochemical approach to the problem of differential quantitative determination of natural zeatin forms is suggested. This approach does not require preliminary separation of experimental samples into individual fractions. True concentrations of zeatin and zeatin riboside in the T. officinale ovary were calculated based on the average values of apparent concentrations of endogenous cytokinins. When the embryo sac maturation had been completed, there was a threefold increase in the zeatin riboside concentration within the following 12 h. By the time of the first division of an unfertilized ovicell (i.e., within the next 12 h), there had been a twofold decrease in the zeatin riboside concentration. Therefore, at early stages of division of the unfertilized ovicell the zeatin riboside concentration virtually returned to the initial level. In contrast to zeatin riboside, there was a steady trend toward an increase in the zeatin concentration in the T. officinale ovary. Within the first 12 h and the next 12 h after completion of the embryo sac maturation, the zeatin concentration was increased 1.5-fold and 2-fold, respectively. The results of this work provide a pioneering insight into the dynamics of various natural forms of zeatin during the reproductive process. The immunochemical approach to quantitative monitoring of various natural forms of zeatin and their dynamics during embryogenesis suggested in this work can be extended to similar biological, medical, and agricultural problems of differential determination of low-molecular-weight agents of similar structure but different biological activity.     

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.     

Effect of some imidazopyrimidines on tobacco callus cultures

Abstract

Several 7-chloro-imidazo(1,2-c)pyrimidines were tested on tobacco callus cultures in order to verify a possible cytokinin and anticytokinin-like activity. These compounds were used alone and in combination with kinetin or zeatin.

The aminofurfuryl derivatives showed a strong inhibitory action on cell multiplication and this effect was enhanced when they were mixed with kinetin or zeatin.

The isopentenyl derivatives on the contrary were able to induce cell division in tobacco callus.     

Identification of Cytokinins of Root Nodules of the Garden Pea, Pisum sativum L

Five cytokinin activities which induced soybean callus proliferation were detected in ethanol extracts of root nodules of the garden pea (Pisum sativum L., cv. Little Marvel). The most active factors among them were identified as zeatin and its riboside on the basis of their mobility on thin layer chromatography in three solvent systems. Smaller activities of zeatin ribotide, isopentenyladenine and its riboside were also detected. Cytokinin activity gradually decreased with the cultivation period, but no qualitative change in the active compounds was found.     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VI. Metabolism of zeatin riboside applied via the tips of nodulated pea roots

[³H]zeatin riboside was supplied in physiological quantities to pea (Pisum sativum L. cv Greenfeast) plants by replacing the root tip with a small vial containing [³H]zeatin riboside, to simulate the normal supply of cytokinin. Radioactivity was transported to the root nodules. Analysis by two-dimensional thin layer chromatography revealed that little³H remained as zeatin riboside in root or nodule tissue at the end of the labeling period (2, 5, or 8 d) and suggested that the following compounds were metabolites of [³H]zeatin riboside: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, nucleotides of adenine and zeatin, and the dihydro-derivatives of many of these compounds. The O-glucosides (and in particular, O-β-D-glucopyranosyl-9-β-D-ribofuranosylzeatin) appeared to be more prominent metabolites in the effective nodules formed by strain ANU897 than in the ineffective nodules produced by strain ANU203. However, no other appreciable differences were detected between effective and ineffective nodules in their metabolism of zeatin riboside. There were few marked differences between root and nodule tissue; however, in some experiments, the nodules contained a higher proportion of O-glucoside metabolites, and generally root tissue contained a greater proportion of zeatin and/or dihydro-zeatin, zeatin riboside and/or dihydrozeatin riboside, adenine and the nucleotides of zeatin and adenine, as metabolites.     

Effects of temperature and gibberellin on the activity of endogenous cytokinin-like substances in leaves of Begonia x cheimantha

Changes in activity of endogenous cytokinin-like substances were examined in intact plants and excised leaves of Begonia x chemantha Everett cv. Prinsesse Astrid (Christimas Begonia) by means of the tobacco callus bioassay. Cytokinin activity in the leaves of intact plants was higher in plants grown at 18°C than in those grown at 21° or 24°C. In excised leaves, an increase in cytokinin activity was observed during the first 4 days following leaf detachment. However, after the seventh day cylokinin activity decreased again. This decrease was more profound in leaves exposed to 24°C than in those exposed to 18°C.
Treatment of detached leaves with gibberellic acid (2.8 m M ) caused an increase in measurable cytokinin activity. This increase was more profound in the zones of activity which correspond with zeatin glucosides on paper and Sephadex LH-20 chromatography. Additional zones of activity appeared after Sephadex chromatography. These were of a more slow moving nature with elution volumes corresponding to N^b-(Δ²-isopentenyl)adenine and its derivaties. Water-treated control leaves had higher activity in the regions corresponding to 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.     

Inhibitors of cytokinin metabolism III. The inhibition of cytokininN-glucosylation in radish cotyledons

Summary Cytokinins are deactivated in radish cotyledons by conversion to 7- and 9-glucosides. In a search for inhibitors of this metabolism, the following compounds were found to be effective: (a) 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine; (b) 3-isobutyl-1-methylxanthine; (c) papaverine; (d) theophylline; (e) caffeine; and (f) theobromine. The order of effectiveness was: (a)>(b)=(c)>(d)=(e)=(f). While the methylxanthines (b) and (d) inhibited formation of both 7- and 9-glucosides of 6-benzylaminopurine (BAP) approximately equally, compounds (a) and (c) preferentially inhibited formation of BAP 9-glucoside. Inhibition of BAP glucoside formation by (a) at 1.3 mM elevated the level of free BAP and BAP nucleotide 23- and 94-fold, respectively. While abscisic acid (ABA) suppressed conversion of zeatin riboside to zeatin 7-glucoside in radish cotyledons, it did not inhibit conversion of BAP to glucosides. Hence, ABA probably does not inhibit the glucosylating enzymes directly but rather reduces the availability of free zeatin when zeatin riboside is supplied. Auxins and nutrient supply did not affect conversion of zeatin riboside to zeatin 7-glucoside. Relative to cotyledons developed in light, those developed in darkness had a reduced capacity to convert zeatin riboside to zeatin 7-glucoside. The results presented have identified types of chemical structures which could be developed to provide more effective inhbitors of cytokininN-glucosylation.For part II in the series Inhibitors of cytokinin metabolism see Zhang et al. (1989).     

Phytohormones,Rhizobium mutants, and nodulation in legumes. VI. Metabolism of zeatin riboside applied via the tips of nodulated pea roots

[³H]zeatin riboside was supplied in physiological quantities to pea (Pisum sativum L. cv Greenfeast) plants by replacing the root tip with a small vial containing [³H]zeatin riboside, to simulate the normal supply of cytokinin. Radioactivity was transported to the root nodules. Analysis by two-dimensional thin layer chromatography revealed that little³H remained as zeatin riboside in root or nodule tissue at the end of the labeling period (2, 5, or 8 d) and suggested that the following compounds were metabolites of [³H]zeatin riboside: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, nucleotides of adenine and zeatin, and the dihydro-derivatives of many of these compounds.The O-glucosides (and in particular, O--D-glucopyranosyl-9--D-ribofuranosylzeatin) appeared to be more prominent metabolites in the effective nodules formed by strain ANU897 than in the ineffective nodules produced by strain ANU203. However, no other appreciable differences were detected between effective and ineffective nodules in their metabolism of zeatin riboside. There were few marked differences between root and nodule tissue; however, in some experiments, the nodules contained a higher proportion of O-glucoside metabolites, and generally root tissue contained a greater proportion of zeatin and/or dihydro-zeatin, zeatin riboside and/or dihydrozeatin riboside, adenine and the nucleotides of zeatin and adenine, as metabolites.     

The Metabolism of Zeatin and Zeatin Riboside by Soya Bean Callus

Five cell division inducing compounds were found in soya beancallus irrespective of whether it wes grown on a zeatin or zeatinriboside containing basal medium. In both cases the major metaboliteseems to be zeatin glucoside. The significance of this metabolicstep in plant tissue is discussed.