Changes in Cytokinins and Gibberellin-Like Substances in Pinus radiata Buds during Lateral Shoot Initiation and the Characterization of Ribosyl Zeatin and a Novel Ribosyl Zeatin Glycoside

Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of “short shoots” (needle fascicles) to “long shoots” (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.     

Genes Encoding Zeatin O-Glycosyltransferases

Zeatin is a highly active and ubiquitous cytokinin. O-Glycosylation of zeatin occurs widely in plant tissues and is likely to be important in regulating the level of active cytokinins. Enzymes mediating the conversion of zeatin to O-glucosylzeatin and O-xylosylzeatin have been isolated from Phaseolus seeds. The corresponding genes have been cloned recently. The current status of molecular and biochemical studies of these genes and enzymes is summarised in this paper.     

Identification of Zeatin and Zeatinriboside in Coconut Milk

Zeatin and zeatinriboside were identified from the milk of mature Cocos nucifera fruits. It would appear as if the two compounds are present in roughly equal proportions. Ethyl acetate extraction in a liquid-liquid extractor at an alkaline pH (8.0) proved to be a very efficient method of extracting zeatinriboside. Partitioning with water-saturated n-butanol proved to be the best way of extracting zeatin.     

6-(gamma,gamma-Dimethylallylamino)Purine As A Precursor of Zeatin

A 6-(γ,γ-dimethylallylamino) purine-like compound was found in the culture medium of Rhizopogon roseolus, which had been shown earlier to synthesize zeatin. The role of 6-(γ,γ-dimethylallylamino) purine as a precursor of zeatin was studied. Rhizopogon was furnished with 6-(γ,γ-dimethylallylamino) purine-8-¹⁴C. Cochromatography, oxidation studies with potassium permanganate, and bromination indicated that labeled zeatin ribonucleoside was isolated from the medium. The fungus also incorporated labeled adenine, hypoxanthine, and 4-amino-5-imidazole carboxamide into zeatin ribonucleoside.     

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.     

The Identification of Zeatin Glucoside from Coconut Milk

By means of chromatographic, mass spectrometric and enzymatic techniques the major cytokinin in butanol extracts from the milk of Cocos nucifera fruits was identified as zeatin glucoside.     

Spatial and Temporal Distribution of Zeatin and Zeatin Riboside during the Period of the Egg-Cell Activity in the Ovaries of Amphimictic and Apomictic Plants

Spatial and temporal distribution of zeatin and zeatin riboside was studied in the ovaries of amphimictic (Triticum aestivum L.) and apomictic (Taraxacum officinale Web.) species during the period from the quiescence release of the egg cell to its first division. This period was divided into three stages: (1) no signs of division of the central cell, (2) division of the central cell and no signs of division of the egg, and (3) division of the egg. Cytokinin content was determined in ovary and its four sections (basal, medial, apical, and ovule) using an original modification of ELISA. Apomicts and amphimicts were shown to be similar in the pattern of changes in the cytokinin content in basal and medial zones but differed substantially in that of apical section and ovule.     

The Metabolism and Translocation of Zeatin in Intact Radish Seedlings

After the roots of intact radish seedlings had taken up [³H]zeatinfor 1 h, the seedlings were transferred to nutrient lackingzeatin and extracted at intervals. After 23 h in the absenceof zeatin, 6 per cent of the radioactivity extracted per seedlingwas recovered from the de-ribbed cotyledon laminae, 4 per centfrom the hypocotyls, and 87 per cent from the roots. Per unitweight of tissue, the radioactivity extracted from the rootwas about 40 times that recovered from any other region. Zeatin was rapidly metabolized by the root tissue, and 4 to9 h after transfer of the seedlings to nutrient lacking zeatin,accounted for a negligible proportion of the radioactivity.Initially zeatin riboside 5'-monophosphate was the principalroot metabolite, but after 9 h, 7-glucosylzeatin (raphanatin)was the dominant metabolite. Conversion of zeatin to dihydrozeatinwas not detected. Raphanatin was also the major metabolite inthe cotyledon laminae where some free zeatin was detectable.The principal metabolites in hypocotyl extracts were AMP andzeatin riboside 5'-monophosphate but zeatin riboside was theonly significant source of radioactivity in the xylem sap. When [³H]zeatin was applied directly to cotyledon laminae, 99per cent of the radioactivity was localized in the treated laminae;however traces of zeatin were detected in the roots. In radish seedlings, zeatin riboside appears to be the translocationalform of zeatin, while raphanatin may be a storage form.     

Zeatin supplement improves scopolamine-induced memory impairment in mice

In this study, our aim was to clarify the ameliorative effects of zeatin, a development hormone in plants. Zeatin mitigated cognitive deficits and showed AChE inhibition in scopolamine (Scop)-induced mice following 21 d of zeatin treatment. After administration of Scop for 30 min, each mouse performed Y-maze and step-down latency tasks as a check on immediate against cognitive function. The results showed that zeatin administration attenuated Scop-induced memory damage and decreased AChE activity in the mice. This suggests that zeatin might be useful for protecting cognitive dysfunction, as well as for reducing the activation of AChE in dementia.     

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.     

Comparative Activity of Isomers of Zeatin and Ribosyl-Zeatin on Funaria hygrometrica

The activities of isomers of zeatin, ribosyl-zeatin, and 6-(γ,γ-dimethylallylamino) purine (i⁶Ade) on the moss Funaria hygrometrica are compared by measuring the ability of the cytokinins to induce callus or gametophores. The cis- and trans-ribosyl-zeatins were inactive, and therefore this kind of bioassay cannot be used as evidence for the presence or absence of a cytokinin in tests on natural products.     

Influence of Endogenous Cytokinins on Reverse Mobilization in Cotyledons of Cicer arietinum L: Reproduction of Endogenous Levels of Total Cytokinins, Zeatin, Zeatin Riboside, and Their Corresponding Glucosides

The embryonic axis plays an essential role in the mobilization of the main reserves of the cotyledons of seeds of Cicer arietinum L. cv Castellana. This control by the axis of the metabolism of the storage products of the cotyledons largely takes place through the cytokinins, which are transported from the embryonic axis to the cotyledons where the mobilization of reserves begins. The principal regulatory role of the endogenous cytokinins concerns the metabolism of carbohydrates and proteins; there is less influence on lipid metabolism. However, each cytokinin seems to have a different role in the mobilization processes. The glucosides, glucosyl zeatin riboside, and glucosyl zeatin act only as storage forms of the hormones. Zeatin riboside affects mainly the mobilization of carbohydrates and has less effect on protein mobilization. Zeatin regulates both the mobilization of carbohydrates and that of proteins and is more marked in the latter case.     

Isolation of Legume Glycosyltransferases and Active Site Mapping of the <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> Zeatin O-glucosyltransferase ZOG1

O-Glycosides of the cytokinin zeatin are found in many plant tissues. They provide protection against degradative enzymes and may serve as cytokinin reserves. Two zeatin glycosyltransferase (GT) genes, an O-glucosyltransferase (ZOG1) from Phaseolus lunatus and an O-xylosyltransferase (ZOX1) from P. vulgaris, were previously isolated. Five novel bean and soybean GT genes with high sequence identity to ZOG1 were isolated, sequenced, and expressed, along with two such genes from rice and one from tomato. None of the recombinant proteins showed GT activity with zeatin. By comparing the ZOG1 sequence to the 3D model of Medicago truncatula UGT71G1, four regions possibly important to zeatin binding were identified, and mutation studies identified one amino acid within each region (R59, D87, L127, and F149) whose mutation strongly impaired enzyme activity. The new bean and soybean GTs differ from ZOG1 in one (PlGT2 and GmGT2) to three (GmGT1) of these residues. Mutation of one such GT (PlGT2) to render it identical to ZOG1 at the four implicated residues conferred low enzyme activity, providing further support for the importance of these amino acids in recognizing zeatin as substrate. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Metabolism of Hormones during Seed Germination and Release from Dormancy: III. The Effects and Metabolism of Zeatin in Dormant and Nondormant Ash Embryos

Zeatin and zeatin-9, β-ribonucleoside enhance the germination of dormant ash embryos. While the first macroscopic signs of germination appear only after about 72 hours, 12 hours of exposure to 50 μm zeatin is as effective as continuous incubation. There must be barriers against transport out of the embryos since 8-¹⁴C-zeatin and its metabolites, zeatin-9, β-ribonucleoside, the 5′-mono and the suspected di- and triphosphates, accumulate against a concentration gradient. Zeatin ribonucleoside is about as effective as zeatin in enhancing embryo germination, yet the internal 8-¹⁴C-zeatin level is lower by a factor of about 50 when the ribonucleoside is fed. The physiological effects of zeatin and abscisic acid on the germination of ash embryos are antagonistic. There is, however, no evidence that abscisic acid has a significant effect on 8-¹⁴C-zeatin uptake or conversions.     

Influence of Zeatin on Flowering in Root Forming Cuttings of Anagallis arvensis L.

Applied zeatin affected flowering in cuttings of Anagallis arvensis(long-day plant). According to the zeatin concentration andthe root formation stage, zeatin inhibited flowering at differentdegrees or slightly promoted it. Thus, the root primordium settingseemed to be correlated with this effect of zeatin. (Received February 14, 1984; Accepted May 18, 1984)     

Changes in Cytokinin Distribution in the Ovule and Ovary of Taraxacum officinale Web. in Early Stages of Embryogenesis

Quantitative changes in the hormonal status of the ovules and ovaries were first studied in Taraxacum officinale Web. at the early stages of embryogenesis. The plant material was analyzed by ELISA using labeled anti-rabbit antibodies. A new procedure for differential and quantitative determination of the main endogenous cytokinins based on the estimation of the effective zeatin and zeatin riboside concentrations from calibration curves constructed using zeatin and zeatin riboside as standard antigens was developed. It was shown that, at the three initial stages of embryogenesis examined, the concentration of zeatin uniformly increased in T. officinale ovules. The concentration of zeatin riboside, conversely, uniformly decreased. However, their total concentration changed insignificantly. A gradual increase in the concentration of the active and storage hormone forms from the ovary to the ovule was shown.     

A new micromethod for differential quantitative assay of zeatin and zeatin riboside

A new method is proposed for differential quantitative assay of two major endogenous cytokinin forms. It is based on determination of two effective parameters-concentrations of zeatin and zeatin riboside--with the use of appropriate antigens as standards. The method can be used for determining cytokinins in small samples of plant tissues without extract fractionation. This study pioneers in quantitation of changes in the hormonal status of ovules and ovaries of Triticum aestivum L. at early stages of embryogeny. A gradual increase in the content of the active and storage forms of the hormones from the ovary to the ovule was revealed.     

Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea L. Crown Gall Tumor Tissue

Recently detected but unidentified cytokinin activity in crown gall tumor tissue from Vinca rosea L. grown on media containing sources of reduced nitrogen has now been attributed to two adenine-type cytokinins. These compounds are glucopyranosyl derivatives of zeatin and ribosylzeatin. The substitution in each case is on the isopentenyl chain of the parent compound. Neither of these compounds had activity in the soybean callus bioassay at concentrations lower than 1 nm whereas zeatin had activity at 0.1 nm.     

Accumulation of zeatin O-glycosyltransferase in Phaseolus vulgaris and Zea mays following cold stress

Zeatin O-glycosides have been reported as inactive and stable storage forms of cytokinins whose concentrations increase in cold stressed plants. Zeatin O-glycosides accumulation in developing bean seeds has been correlated with an increase of zeatin O-glycosyltransferase , which is specific to trans-zeatin, and catalyzes the conjugation of zeatin O-glycosides. When Phaseolus vulgaris and Zea mays seedlings were grown for 3 days at 25 and then incubated at 4 or 10 for 6 days no further growth was observed in roots. Hypertrophy was observed in the root tips of both species. In shoot-hypocotyl complexes, in contrast, growth occurred when seedlings were incubated at 10 . Western analysis, with Mabs specific to zeatin O-glycosyltransferase, detected antigenically related proteins in roots, shoot tips and cotyledons after seedlings were cold stressed for 1–6 days at 4 or 10 . Immunolocalization, of both maize and bean root sections grown at 25 revealed antigenically related proteins that were detected at low levels in cortical cells. The signal intensified upon cold stress. The localization of zeatin O-glycosyltransferase in Z. mays root tips was directly comparable to the distribution of the zeatin O-glycosides. The enzyme was detected in the nucleus, cytoplasm, and closely associated with the plasma membrane and in the cell wall of Z. mays root cells. Southern analysis suggested that more than one gene in Z. mays that were homologous to zeatin O-glycosyltransferase in P. vulgaris. Zeatin O-glycosyltransferase may be involved in modulation of cytokinins under cold stress.     

Chromatography of Cytokinins on a Neutral Polystyrene Resin: A Simple Procedure for the Separation of the cis and trans Isomers of Zeatin or Ribosylzeatin

A simple procedure for the separation of the cis and trans isomers of zeatin and ribosylzeatin by column chromatography on a neutral polystyrene resin, Porapak Q, in aqueous ethanol solutions is reported. The method has been used to examine the stereoisomer composition of ribosylzeatin isolated from wheat germ transfer RNA. Chromatographic data for several other cytokinins are also presented.