Variations in the Levels of cis- and trans-Ribosylzeatins and Other Minor Cytokinins during Development and Growth of Cones of the Hop Plant

The endogenous cytokinins in cones of the hop plant (Humuluslupulus L. cv. Shinshuwase) were identified by combined gaschromatography and selected ion current monitoring (GC-SIM)and high performance liquid chromatography as ribosyl-cis-zeatin,ribosyl-trans-zeatin, cis-zeatin, trans-zeatin and ribosyl-trans-zeatin-O-glucoside.The contents of these cytokinins in both fertilized and unfertilizedcones at various growth stages were determined using GC-SIMand/or bioassay. Based on these data, the rapid growth of thefertilized cone is attributed mainly to ribosyl-trans-zeatinwhich accumulates mostly in the seed. Ribosyl-cis-zeatin wasfound in both fertilized and unfertilized cones. In the latter,ribosyl-cis-zeatin was quantitatively a major cytokinin andseemed to cooperate with ribosyl-trans-zeatin and trans-zeatinin promoting the growth. (Received January 12, 1981; Accepted February 10, 1981)     

Identification of N6-(3-methyl-but-2-enyl) adenosine, zeatin, zeatin riboside, gibberellin A19 and abscisic acid in shoots of the hop plant

Young shoots of the hop plant (Humulus lupulus L.) have beenfound to contain at least three cytokinins, one gibberellin,and abscisic acid. Two of the cytokinins were identified aszeatin riboside and N⁶-(3-methyl-but-2-enyl)adenosine (2iPA)based on gas chromatography-mass spectrometry (GC-MS) afterseveral purification steps. Another cytokinin was assumed tobe zeatin from its chromatographic behavior. Gibberellin A₁₉was identified by GC-MS and mass fragmentography, and ABA characterizedby GC-MS. (Received March 30, 1978; )     

Isolation and Identification of L-Pipecolic Acid and Nicotinamide as Flower-Inducing Substances in Lemna

Flower-inducing factors in extracts of flowering Lemna gibbaG3 were investigated using Lemna paucicostata 151 as the bioassayplant. Fractions with flower-inducing activity were obtainedafter several purification steps. Two of the active substanceswere identified as L-pipecolic acid and nicotinamide by MS andNMR analyses. Both L-pipecolic acid and nicotinamide exhibited flower-inducingactivity in L. paucicostata 151 grown on one-tenth-strengthM medium containing benzyladenine, the former being ten timesas active as the latter. L-Pipecolic acid was active even at0.01 ppm (7.8 ? 10^–8 M). The effect of L-pipecolic acidon flowering strongly depended upon the presence of exogenouscytokinin. The coexistence of cytokinin seemed to be essentialfor L-pipecolic acid to exhibit flower-inducing activity. Incontrast, the effect of nicotinamide on flowering was basicallythe same as that of benzoic acid or nicotinic acid. (Received February 9, 1987; Accepted May 21, 1987)     

Cytokinin translocation: Changes in zeatin and zeatin-riboside levels in the root exudate of tomato plants during their development

Summary The zeatin and zeatin riboside content of tomato (Lycopersicon esculentum Mill.) root exudates were determined at different stages of development. Zeatin riboside was found to be the major translocational form of cytokinin in the xylem during early vegetative growth. During flower bud formation this cytokinin decreased markedly in concentration so that, at anthesis, there was no appreciable difference in the zeatin and zeatin riboside concentration in the root exudate.     

Comparison of zeatin indoleacetate with zeatin and indoleacetic Acid in the tobacco bioassay

Zeatin indole-3-acetate, 6-[4-(indole-3-acetoxy)-3-methyl-trans-2-butenylamino]purine, is at least as effective as zeatin on a molar basis in satisfying the cytokinin requirement for growth and bud formation in tobacco bioassays. It is less effective than indole-3-acetic acid and is needed as a variable function of the cytokinin concentration for satisfying the optimal requirement of an auxin. Comparisons of the types of growth and yield of tissue obtained with serial concentration of the ester and with equimolar mixtures of its free base and acid indicate that the relative requirement for auxin changes with the concentration of cytokinin and is related to the types of callus growth and differentiation which occur. The results also suggest that the ester serves as a source of auxin only after modification, presumably by hydrolysis to indoleacetic acid.     

The Effect of Cytokinins on Vitrification in Melon and Carnation

The influence of exogenous cytokinin on vitrification of melonand carnation buds grown in vitro was assessed. In melon, theanatomical traits of vitrification were found to be similarto those previously described for carnation grown on a highconcentration of auxin. In carnation, the plantlets treatedwith high cytokinin concentrations became bushy but the anatomicalstructure remained normal. The relation of cytokinin and vitrificationis discussed. Cucumis melo L., Dianthus caryophyllus L., vitrified plantlets, habituation, cytokinin     

Quantitation of cytokinins in biological samples using antibodies against zeatin riboside

The cross-reactivity of antibodies elicited in rabbits against zeatin riboside, to a wide range of naturally occurring cytokinins, was examined. As well as to zeatin riboside, the antisera cross-reacted to a considerable extent with zeatin, lupinic acid, zeatin-9-glucoside, zeatin riboside 5′-monophosphate and to a much lesser, but measurable extent, with dihydrozeatin riboside and dihydrozeatin. Chromatographic methods were devised which allowed separation of all these cross-reactive compounds. Four biological samples, extracts of immature Zea mays kernels, immature seeds of Lupinus luteus, and Datura innoxia crown gall tumor tissue, and a sample of Agrobacterium tumefaciens culture supernatant, were purified by these chromatographic methods, using [³H]zeatin riboside as a recovery marker, and at each stage of the purification process, were subjected to radioimmunoassay over a range of dilutions. At each stage of sample purification, sample dilution curves were found to be parallel to the standard curve. Sample cytokinin levels estimated by radioimmunoassay were in close agreement to those available in the literature for similar samples assayed by alternative methods. However, in some samples, unknown cross-reacting compounds were detected.     

Control of Morphogenesis in Sorghum by 2,4-Dichlorophenoxyacetic Acid and Cytokinins

2, 4-Dichlorophenoxyacetic acid caused a shortening of rootsand shoots when mature seeds of Sorghum bicolor (L.) Moenchcv. BT3197 were germinated on Murashige and Skoog (MS) mediumthat contained 2,4-D. Shoot growth was restored with cytokinins.A callus formed at the nodal region, the further differentiationof which was determined by the ratio of 2,4-D and cytokininsin the initial culture medium. A high auxin to cytokinin ratiopromoted primarily root differentiation while a high cytokininto auxin ratio promoted multiple bud development. Isolated shootapical meristem with the subtending node produced embryogeniccallus at low cytokinin levels and green buds on high cytokininlevels when cultured in the presence of 2,4-D. It is concludedthat cells potentially capable of differentiation into roots,somatic embryos or axillary buds are present in the first nodalregion. Sorghum bicolor, organogenesis, embryogenesis, 2, 4-D: cytokinin ratios, tissue culture     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)     

Types, Formation, and Metabolism of Cytokinins in Leaves of Alnus Glutinosa (L.) Gaertn.

The nature of the cytokinins present in mature, expanded leavesof alder (Alnus glutinosa (L.) Gaertn.) was investigated usinga chromatographic system capable of partially resolving zeatinfrom (?)-dihydrozeatin. A zeatin-like cytokinin, present asboth a ‘free’ form and as a polar conjugate, postulatedto the the O-ß-D-glucoside of zeatin, accounted forthe bulk of the cytokinin activity (detected by the soybeancallus assay). Studies on the fate of [8-¹⁴C]zeatin supplied to detached alderleaves indicated this cytokinin to undergo extensive metabolism.An appreciable proportion of the radioactivity was incorporatedinto 80% methanol-insoluble and soluble acidic/neutral fractions,while adenosine- and adenine-like peaks were prominent metabolitesin the basic n-butanol-soluble fraction. Small amounts of glucosides,with the properties of both zeatin-O-ß-D-glucosideand dihydrozeatin-O-ß-D-glucoside were formed, thelatter becoming the most prominent with time. The ability of alder leaves to form glucoside directly from(?)-dihydrozeatin was assessed using the soybean callus assay.(?)-Dihydrozeatin was subject to a relatively rapid, continuousand substantial conversion to its putative O-ß-D-glucosideand cytokinin activity was consequently conserved while, incontrast, leaves supplied with zeatin exhibited a progressiveloss of cytokinin activity and, in agreement with the radioactivityexperiments, produced only a small amount of activity attributableto the putative O-glucosides. The significance of the observed cytokinin metabolism is discussedin relation to the endogenous cytokinin status of the leaf.     

Isolation of zeatin riboside from the chicory root

Summary From 250 kg of fresh chicory roots about 2 mg of a crystalline cytokinin were obtained. This substance was identified as ribosylzeatin (trans isomer). From the procedure employed it seems unlikely that the isolated cytokinin comes from the degradation of tRNAs; rather, it may constitute a separate pool of cytokinins.     

Hormonal and Structural Aspects of Fruit Development in the Orchid, Epidendrum

Endogenous cytokinin and gibberellin-like activity were measuredby bioassay in developing fruit of the orchid Epidendrum ibaguense.Cytokinins decline during the first 30 d after pollination,then begin to accumulate, with very high levels (1–13µ g zeatin eq. g^–1 dry wt. ) in the mature fruitand seed. The major structural change in developing fruit duringthe first 30 d is the ongoing cell division in the fruit wall.By day 30 most ovules have been fertilized and embryo developmentbegins. The increase in cytokinin activity thus coincides withthe onset of embryo development. Gibberellin levels declinein the fruit throughout development, although high activity(0.9 µ g GA₃ eq. g^–1 dry wt. ) is observed in themature seed. The mature embryo shows no obvious structural differentiationinto embryonic axis and cotyledon and no endosperm develops.     

A Comparative Auxin and Cytokinin Study in Normal and to-2 Mutant Tomato Plants

Torosa-2 (to-2), a tomato mutant with strong apical dominance,was studied in order to determine the mechanism of shoot outgrowthcontrol. In decapitated or defoliated to-2 plants only a fewshoots grew and IAA or morphactin application had little oronly short term effects. No differences were found in auxinand cytokinin activities between normal and mutant plants upto 20 days after sowing. In the period from 40 to 90 days theIAA content increased equally in both genotypes. During thesame period, however, cytokinin increased only in normal plants.The results obtained with the to-2 mutant indicate that budsdo not shoot, probably because bud differentiation does notoccur. The ratio of auxin to cytokinin clearly was affectedby the low level of cytokinin in all tissues of the to-2 plants.This led us to the conclusion that insufficient quantities ofcytokinin for lateral bud differentiation is the cause of thestrong apical dominance in to-2. (Received January 20, 1982; Accepted April 26, 1982)     

Exogenous zeatin accumulation in wheat-root cells and its role in regulation of cytokinin transport

It was shown that the cytokinin content in the xylem sap of a wheat plant treated with exogenous zeatin was about ten times lower than in the nutrient solution in 24 h. Cytokinins were accumulated in roots rather shoots of treated plants. These data demonstrate the existence of a barrier in the cytokinin pathway from the nutrient solution to plant shoots. The deposition of lignin and suberin in stele detected with Sudan @III is enlarged with an increase in the distance from the tip of the root. The augmented content of suberin and lignin coincided with reduced cytokinin immunolabeling in root cells revealed by monoclonal antibodies to cytokinin and secondary gold-labeled antibodies. The accumulation of exogenous cytokinin in root stele cells shows that Casparian bands are not the only barrier on the cytokinin pathway to plant shoots. Intensive cytokinin immunolabeling in parenchyma cells surrounding stele vessels indicates the accumulation of cytokinin by these cells and suggests that there are mechanisms that limit the hormone loading in xylem vessels during transport to the shoot. The role of cytokinin transporters in this process is discussed.     

Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.     

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     

Biosynthesis of Cytokinin in Germinating Seeds of Zea mays

The embryos of germinating Zea mays seed were supplied with[¹⁴C]-adenine Following incubation, the tissue was extractedand extensively purified by non-exchange chromatography andthin layer chromatography. Radioactivity was found to be incorporatedinto zeatin nucleotide indicating that the embryo in the germinatingseed is capable of cytokinin biosynthesis. Key words: Zea mays cytokinin, zeatin nucleotide, biosynthesis, seed     

Cytokinin Regulation of Flower and Pod Set in Soybeans (Glycine max(L.) Merr.)

Exogenous application of cytokinin to raceme tissues of soybean(Glycine max(L.) Merr.) has been shown to stimulate flower productionand to prevent flower abortion. The effects of these hormoneapplications have been ascertained for treated tissues, butthe effects of cytokinins on total seed yields in treated plantshave not been evaluated. Our objectives were to examine theeffects of systemic cytokinin applications on soybean yieldsusing an experimental line of soybeans, SD-87001, that has beenshown to be highly sensitive to exogenous cytokinin application.Soybeans were grown hydroponically or in pots in the greenhouse,and 6-benzylaminopurine (BA) was introduced into the xylem streamthrough a cotton wick for 2 weeks during anthesis. After theplants had matured, the number of pods, seeds per pod, and thetotal seed weight per plant were measured. In the greenhouse,application of 3.4 x 10^-7 moles of BA resulted in a 79% increasein seed yield compared with controls. Results of field trialsshowed much greater variability within treatments, with consistent,but non-significant increases in seed number and total yieldsof about 3%. Data suggest that cytokinin levels play a significantrole in determining total yield in soybeans, and that increasingcytokinin concentrations in certain environments may resultin increased total seed production. Copyright 2001 Annals ofBotany Company Glycine max, soybean, flower abortion, cytokinin, 6-benzylaminopurine, hydroponic, seed yield, wicking     

Involvement of tRNA Bound Cytokinin on the Gall Formation in Zizania

To understand the role of the conjugated forms of cytokininin gall formation, tRNA was extracted and purified from vegetativeand flowering shoots of Zizania, then hydrolysed by RNase T,or acid to release the conjugated forms of cytokinin. The resultsindicated that the level of cw-zeatin riboside fromZizania galltissue was 3 to 5 times higher than that of flowering Zizania,and the cytokinin activity in terms of kinetin equivalence was1.6 times higher in Zizania gall tissue than that of floweringZizania. It was found that higher amounts of cytokinin alsopromote protein synthesis. Due to the difference in levels ofthe conjugated forms of cytokinin between vegetative and floweringZizaniashoots, it is implied that the conjugated form of cytokininmay play an important role during the process of gall formationin Zizania latifolia. Key words: Gall formation, cytokinin, Zizania latifolia, RNA, smut fungus, water oat