The Formation, Isolation, and Biological Activity of a Cytokinin 7-Glucoside

Concentrations of 10-8 to 10-5 M O-B-D-glucopyranosylzeatin are less active than zeatin and zeatin riboside in the soybean (Glycine max L.) callus bioassay. At a concentntion of 10-4 M the glucoside was, bowever, more active or alternatively le toxic than smilar concentrations of zeatin and zeatin riboside. Appl zeatin-O-lucoside is readly metabolzed by soybean callus and both zeatin and zeatin riboside could be extracted from callos grown on basal medium contaiing the 0-glucoside.The occurrence of cytokinin glucosides in plants is now well established (6, 12, 15, 16, 18, 20). When zeatin (6,7,10,17) and zeatin riboside (17) are applied to plant material they are rapidly converted to their respective glucosides. The exact physiological function of the endogenous cytokinin glucosides is presently not known. It has been suggested that they are storage or transport forms of the cytokinins (10,11,14) or that they represent attempts by plant tissue to inactivate or bind these physiologically active compounds (6). In a number of cases (15,16) it has been reported that soybean callus responds better to cytokinin glucosides than to their free bases obtained after hydrolysis with f-glucosidase. Whether this means that the cytokinin glucosides represent active forms of the cytokinins (4) and are thus more active in the soybean callus bioassay, or that they are indeed storage (bound, inactive) forms which are less toxic, yet readily available due to hydrolysis as and when required, remains to be determined. The activity of synthetic trans-0-f-D-glucopyranosylzeatin was compared with that of zeatin and zeatin riboside in the soybean callus bioassay. MATERIALS AND METHODSCytokinin-requiring soybean (Glycine max L.) callus, cultured on kinetin, was used in this investigation as it has been shown to contain no free endogenous cytokinins (17). The effect of autoclaving and ,-glucosidase treatment (16) on the synthetic zeatin-O-glucoside was determined by passing untreated, autoclaved and enzyme-treated aliquots (10 ml of 10-4 M solution) through a Sephadex LH-20 column (96 x 2.5 cm) and eluting them with 35% ethanol (1). Forty-ml fractions were collected, dried in 50-ml Erlenmeyer flasks in a stream of air, and then assayed simultaneously for cytokinin activity.To determine the concentration effect on soybean callus, three pieces of callus (20 mg) were transferred to flasks containing serial dilutions (10-4-10-8 M) of zeatin, zeatin riboside (Calbiochem), or trans-O-,&D-glucopyranosylzeatin (for synthesis see below), and then cultured as previously described (9). Experiments were repeated three times with three replicates in each experiment.The C.S.I.R., Pretoria provided financial assistance.To obtain some information as to whether or not zeatin-Oglucoside is metabolized in soybean callus, callus was grown on basal medium containing 10-5 M of this glucoside for 3 weeks. The callus obtained was homogenized with sufficient ethanol to give a final dilution of 80% and extracted at 5 C overnight. The extract was then filt...

Section: Methodsmentioning

confidence: 53%

mentioning

confidence: 99%

Biological Activity of O-β-d-Glucopyranosylzeatin

Staden¹,

Papaphilippou²

1977

“…regulator taken up because of the conversion of the biologically active molecules into derivatives of unknown activity or no activity at all (1, 6,8,9,12,23,24). The cytokinin conjugates formed are glucosides, ribosides, and ribotides.…”

mentioning

confidence: 99%

Cytokinins and Flower Bud Formation in Vitro in Tobacco

Krieken¹,

Croes²,

Smulders³

et al. 1990

Explants from flower stalks of Nicotiana tabacum L. were cultured on different cytokinins to induce flower bud formation. All cytokinins tested except zeatin and zeatin-riboside induced the same maximal number of flower buds. Benzyladenine, benzyladenosine, and dihydrozeatin were the most active compounds whereas isopentenyladenosine and isopentenyladenine acted at a 20-fold higher concentration. These data suggest that the active cytokinins bind to the same receptor with different affinities. The presence of benzyladenine in the medium was necessary only during the first 2 days of culture (initiation period). The equilibrium between benzyladenine and its conjugates (the riboside, glucoside, and nucleotides) after a 4-day pulse was independent of the benzyladenine concentration whether it was inductive or noninductive for bud formation. The level of all derivatives was proportional to the benzyladenine concentration in the medium. Isopentenyladenine was used as a competitive inhibitor of benzyladenine conjugation. Isopentenyladenine concentrations that were too low for bud formation led to a synergistic increase in bud number when applied together with benzyladenine. Isopentenyladenine decreased benzyladenine uptake and conjugation. In spite of the lower uptake, the concentration of free benzyladenine inside the explants was higher in the presence of isopentenyladenine than in its absence whereas the concentration of the 7-glucoside of benzyladenine was lower. It was concluded that the free cytokinin base is the main active compound.The regeneration of organs in small explants from plant tissues offers the possibility to study differentiation on tissues spatially isolated from the whole plant. In this way the qualitative and quantitative response to auxins and cytokinins involved in the regulation of developmental processes can be studied. In tobacco, both the effects of the concentrations and of the ratios of these plant growth regulators on the induction and frequency of roots and/or shoots ( 16) or flower buds (20) have been described. In attempting to understand the mechanism of action of these plant growth regulators, it is important to relate the number of regenerated organs to the actual endogenous auxin and cytokinin concentrations. These concentrations cannot be determined from the amount of growth ' Present address: Department of Botany, University of Guelph, Guelph, Ontario N1G 2W1, Canada. 565regulator taken up because of the conversion of the biologically active molecules into derivatives of unknown activity or no activity at all (1,6,8,9,12,23,24). The cytokinin conjugates formed are glucosides, ribosides, and ribotides. Generally, the glucosides are considered stable and inactive. In contrast, the free base and perhaps also the ribosides and ribotides are active (1 1, 13).Little research has been performed on cytokinin metabolism in relation to developmental processes in plants. Van der Krieken et al. (22) (18), is very well suited to study cytokinin metabolism in relation to flower b...

“…Deleuze et al (9) isolated and identified 7-glucosylbenzyladenine from metabolites of benzyladenine in potato tissue and subsequently assumed that the metabolite from soybean cells was the same on the basis of identical RF values in some solvents (e.g. butan-l-ol-acetic acid-water, 4:1:2, v/v/v) (17). The major metabolite of benzyladenine from soybean cells runs considerably slower than 7-glucosylbenzyladenine in butanol-ammonia (RF 0.25 on 3MM paper in the system used in this report).…”

Section: Resultsmentioning

confidence: 99%

Analysis of Variability in the Amaranthus Bioassay for Cytokinins

Elliott¹

1979

The use of only the upper part of the hypocotyl and the cotyledons in the Amaranthus trcolor bioassay for cytokinins, instead of the whole seedling, was found to reduce the endogenous response and give a higher benzyladenine-dependent response. There is no marked difference in the uptake or metabolism of benzyladenine in whole seedlings compared with that in excised cotyledons.Analysis of variability in the bioassay showed that water availability to the cut seedlings and to whole seedlings is a major factor in the amounts of betacyanin accumulated during the subsequent induction period. The increase in the amount of betacyanin accumulated in response to benzyladenine, following conditions of water stress, is not correlated with differences in benzyladenine uptake. Endogenous production and fusicoccin stimulation is also increased following water loss by cut seedlings. Possible explanations for this stress induction may be found in responses of active transport to changes in turgor pressure.Although pretreatment of the roots of seedlings with mannitol stimulated subsequent induction by excised cotyledons, the presence of mannitol during the induction period inhibited the accumulation of betacyanin. This inhibition is not due to any effect on benzyladenine uptake. The susceptibility of amino acid uptake and polysome profiles to water stress suggests that the inhibition of betacyanin synthesis, a process dependent on protein synthesis, may be due to inhibition either of precursor (tyrosine) uptake or of the synthesis or activity of some enzyme in the pathway.