Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status

The effect of nitrogen on the cytokinin relations of Urtica dioica, the stinging nettle, has been investigated. The plants were grown in quartz sand and nutrient solutions providing levels of nitrate ranging from 1 to 22 mM. Nitrogen supply did not affect biomass production within the range of 3–15 mM NO ₃ ^- . However, the shoot: root ratio of biomass was significantly higher at 15 mM (standard plants) than at 3 mM (low-nitrogen plants) nitrate supply. The cytokinin patterns of the roots, stems and adult, as well as meristematic leaves of plants grown at these two levels of nitrate supply, were determined by means of high-performance liquid chromatography (HPLC) and immunoassays. Enzyme-linked immunosorbent assays (ELISAs) for zeatin riboside, dihydrozeatin riboside, isopentenyladenosine, benzyladenosine and o-hydroxybenzyladenosine enabled the quantification of 17 cytokinins, 13 of which were found in the various tissues of Urtica. trans-Zeatin and its conjugates were the predominant cytokinins in all examined samples. While the free base trans-zeatin and its O-glucoside were the major cytokinins in adult leaves, trans-zeatin riboside was prominent in the other tissues of at least the standard plants. Glucosides of the trans-zeatin type cytokinins were present only in lower amounts. However, considerable amounts of a compound, tentatively identified as cis-zeatin riboside-O-glucoside, were found, particularly in roots and meristematic leaves. Comparatively high amounts of trans-zeatin nucleotide as well as isopentenyladenosine phosphate were also demonstrated in these tissues. Analysis of the root-pressure exudates similarly showed trans-zeatin riboside and, at a lower concentration, trans-zeatin to be the only substantial components. In the low-nitrogen plants, shortage of nitrogen was manifest only in the roots; the nitrogen contents of the shoots did not respond to the nitrogen supply. Likewise, the total content of cytokinins in the shoots of the low-nitrogen plants equaled that of the standard-plant shoots, while it was lower by about 25% in the roots of the low-nitrogen plants. In the latter, the amounts of cytokinins exuded via the root-pressure fluid were also approximately 25% lower. Since the levels of only the trans-zeatin cytokinins in the roots showed a linear correlation with the shoot-to-root ratios, these cytokinins may play an important role in biomass partitioning in Urtica dioica.Abbreviations DHZ dihydrozeatin - ELISA enzyme-linked immunosorbent assay - -G glucoside - HPLC high-performance liquid chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - -N nucleotide (ribotide) - -OG O-glucoside - -R riboside - S/R shoot-to-root (ratio) - Z zeatin This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the SFB 137. The authors wish to thank Mrs. A. Fischbach for skilful technical assistence and Dr. Paul Ziegler (Lehrstuhl für Pflanzenphysiologie, University of Bayreuth, FRG) for linguistic suggestions.     

Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne

The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N ⁶-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N ⁶-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.     

Cytokinin Differences in In Vitro Cultures and Inflorescences from Normal and Mantled Oil Palm (Elaeis guineensis Jacq.)

The mantled abnormality phenotype of the oil palm affects fruit development and thus jeopardizes oil yield. Cytokinins have been implicated in the development of the mantled phenotype. Endogenous cytokinin levels in the normal and mantled phenotypes were compared to determine whether levels of specific cytokinins are associated with mantling. Endogenous cytokinins were identified and quantified in in vitro cultures and inflorescences from normal and mantled oil palms. Twenty-two isoprenoid cytokinins, comprising the zeatin, dihydrozeatin, and isopentenyladenine types, were quantified. Total cytokinin levels, particularly of trans-zeatin and isopentenyladenine types, increased during the in vitro culture process, with the highest levels detected at the proliferating polyembryoid stages. The cytokinins were present mainly in their inactive 9-glucoside forms during in vitro culture. On the other hand, the predominant trans-zeatin cytokinins in inflorescences were present mainly in their ribotide forms, suggesting a metabolic pool of cytokinins for conversion to biologically active free bases or ribosides. Levels of specific cytokinins were significantly different in tissues at different stages. Mantled developed inflorescences contained higher levels of isopentenyladenine 9-glucoside compared with normal inflorescences. Mantled-derived callus tissues had higher isopentenyladenine levels but significantly lower levels of trans-zeatin 9-glucoside, dihydrozeatin riboside, and dihydrozeatin riboside 5′-monophosphate cytokinins compared with normal-derived callus. It would be of considerable interest to verify these specific cytokinin differences in more callus cultures and clones.     

Cytokinins in shoots of the chestnut tree

Five cytokinins, trans-zeatin, 9-β-d-ribosyl-trans-zeatin, O-β-d-glucosyl-trans-zeatin, N⁶-(3-methyl-but-2-enyl)adenine and N⁶-(3-methyl-but-2-enyl)adenosine were identified in shoots of the chestnut tree.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Partial Characterisation of Cytokinins from Root Nodules of Alnus glutinosa (L.) Gaertn

The nature of the substances responsible for the major cytokininactivity in extracts of Alnus glutinosa (L.) Gaertn. root noduleswas investigated by means of chromatographic, chemical, andenzymic methods. Five cytokinins were demonstrated and a furthertwo compounds were probably present in trace amounts. The propertiesof the cytokinins were consistent with their being identicalor closely similar to trans-zeatin, trans-zeatin riboside, zeatin-O-ß-D-glucoside,and a ß-D -glucoside of zeatin riboside together withcertain of the corresponding dihydrozeatin compounds. The greatestpart of the cytokinin activity was represented by the glucosides.     

Monoclonal Antibodies to Plant Growth Regulators: III. Zeatinriboside and Dihydrozeatinriboside

Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed.     

Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA

Application of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) for the analysis of cytokinins was examined. The fragmentation of cytokinins was studied using authentic trans-zeatin (t-Z), trans-zeatin riboside (t-ZR), isopentenyl adenine (i⁶Ade), isopentenyl adenosine (i⁶Ado), benzyl adenine (BAde), benzyl adenosine (BAdo), and kinetin. These cytokinins were effectively ionized by APCI in aqueous acetonitrile. t-Z, i⁶Ade, BAde, and kinetin showed prominent quasi-molecular ions of [M + H]⁺, and ribosylcytokinins clearly showed both [M + H]⁺ and a characteristic fragment ion ([M + H-ribose]⁺), giving some information about their structures. The qualitative and semi-quantitative analyses of cytokinins by LC/APCI-MS were validated in combination with enzyme-linked immunosorbent assay (ELISA) through the analysis of t-ZR in the teratoma of Nicotiana tobacum. t-ZR was conclusively identified and a semi-quantitative estimate of its endogenous levels were provided by the combination of LC/APCI-MS and ELISA. The quantified values obtained by LC/ APCI-MS (single ion detection) and ELISA are in close agreement.     

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.     

Plant Hormones Isolated from “Katahdin” Potato Plant Tissues and the Influence of Photoperiod and Temperature on Their Levels in Relation to Tuber Induction

Qualitative and quantitative analyses were carried out on vegetative tissues of potato (Solanum tuberosum cv. “Katahdin”) in search of natural products thought to play a role in tuber induction. Tissues were obtained from plants initially grown in a growth chamber under noninducing conditions (30°C day and 28°C night with an 18-h photoperiod), and then half of the plants were moved to inducing chambers (28°C day and 13°C night with a 10-h photoperiod) for 10 days prior to tissue harvest. Plants from each chamber were then harvested at 2-day intervals for 10 days, separated into above- and belowground portions, and the lyophilized tissues were extracted and subjected to rigorous purification and separation using high-performance liquid chromatography. This was followed by identification and quantification using combined gas chromatography-mass spectrometry. Compounds isolated and identified included gibberellic acid; cytokinins cis-zeatin riboside, trans-zeatin, trans-zeatin riboside, and isopentenyladenine; and jasmonates jasmonic acid, tuberonic acid and its methyl ester, methyl 7-isocucurbate, and 9,10-dihydromethyljasmonate. Methyl 7-isocucurbate and 9,10-dihydromethyljasmonate were detected for the first time in potato tissue as endogenous compounds. Cytokinin and jasmonate levels generally increased under inducing conditions, whereas gibberellic acid levels declined progressively during the 10-day sampling period. Only gibberellic acid, jasmonic acid, and cis-zeatin riboside levels were significantly influenced by induction.     

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

The effect of cytokinins on growth and sexual organ development in the gametophyte of <Emphasis Type="Italic">Blechnum spicant</Emphasis> L.

In this study, we report the role of exogenous and endogenous cytokinins on growth and sexual organ development in the fern Blechnum spicant L. Spore-derived gametophytes (SG) were cultured in full-strength Murashige and Skoog (1962) liquid medium supplemented with (a) 4.44 μMN⁶-benzyladenine (BAP), (b) a crude extract from mature female gametophytes, and (c) 4.44 μM BAP in combination with the crude extract from mature gametophytes, respectively. Both BAP and the crude extract delayed the gametophyte development, and this effect was increased when they were added together. With respect to sexual organ development, BAP inhibited the sexual organ formation, while the crude extract favored antheridia formation; however, when added together, the percentage of antheridia decreased. The endogenous level of the cytokinins cis-zeatin (cZ), cis-zeatin-riboside (cZR), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), isopentenyl-9-glucoside (iP9G), trans-zeatin (tZ), and trans-zeatin riboside (tZR) were analyzed in female and male gametophytes of B. spicant L. The endogenous levels of cytokinins tZ, cZ, DHZ, cZR, iP, and iPR were higher in female gametophytes than in male gametophytes, with the endogenous iP and iPR content being increased more than 300 and 400 times, respectively.     

Cytokinins in immature seeds of Dolichos lablab

Five cytokinins, trans-zeatin, 9-β-d-ribofuranosyl-trans-zeatin, 9-β-d-ribofuranosyl-cis-zeatin, 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)purine and 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine were identified from immature seeds of Dolichos lablab.     

The development of monoclonal antibodies against the cytokinin zeatin riboside

Seven monoclonal anti-zeatin riboside antibodies were characterized by radioimmunoassay (RIA) and found to measure femtomole (10^?15 M) quantities (～20 pg) of this cytokinin. The antibodies had different measuring ranges defined by the linear portion of the logit/log plots; slopes and intercepts of the line varied considerably between the antibodies. Competitive binding trials againstcis-zeatin riboside (cZR), dihydrozeatin riboside (diHZR), zeatin (Z), and isopentenyl adenosine (iPA) showed differences among the seven antibodies in their cross-reactivities towards these structurally related cytokinins. It was possible to combine selected antibodies to provide a mixture with a predictable measuring range and cross-reactivity; the ability to prepare a highly specific reagent in this manner with well-defined reactivity was noted and differences between monoclonal antibody and polyclonal antiserum probes for measurement of cytokinins were discussed.     

Morphology of potato (Solanum tuberosum L. cv. Sante) stem node cultures in relation to the level of endogenous cytokinins

Stem node culture of the potato (Solanum tuberosum) cv. Sante was used to examine the phenotypical alterations due to different levels of endogenous cytokinins. The altered phenotype, which dramatically deviates from the control phenotype, was induced after treatment of plantlets with 1 m jasmonic acid. Plantlets grown on the medium supplemented with jasmonic acid were taller, with well developed root systems, expanded leaves, thickened stems, and they showed hyperhydric symptoms. Their cytokinin content was about half that of the control plantlets. Morphologic characteristics corresponding to transgenic plants that overproduce cytokinins, including release of axillary buds and inhibited rooting, correlated with the high cytokinin levels in control plants.Abbreviations JA jasmonic acid - Z trans-zeatin - ZR trans-zeatin riboside - ZRMP zeatin riboside 5-monophosphate - Z-9-G trans-zeatin N-9-glucoside - DHZ dihydrozeatin - DHZR dihydrozeatin riboside - DHZRMP dihydrozeatin riboside 5-monophosphate - DHZ-9-G dihydrozeatin 9-glucoside - iP iso-pentenyladenine - iPA iso-pentenyladenosine - iP-9-G iso-pentenyladenine 9-glucoside - HPLC high performance liquid chromatography     

Radioimmunoassay for quantifying the cytokininscis-zeatin andcis-zeatin riboside and its application to xylem sap samples

An antiserum against the cytokinincis-zeatin riboside was raised in rabbits and characterized for use in radioimmunoassays. Cross-reactivity studies demonstrated the specificity of the selected antiserum forcis-zeatin riboside andcis-zeatin in preference to a range of cytokinins and other purines. HPLC systems were developed that separatedcis-zeatin andcis-zeatin riboside from zeatin/dihydrozeatin and zeatin riboside/dihydrozeatin riboside, respectively. These systems enabled the separation of these compounds in xylem sap samples of wheat and oats and their quantification using radioimmunoassay. A TLC system for the separation ofcis-zeatin andcis-zeatin riboside from zeatin/dihydrozeatin and zeatin riboside/dihydrozeatin riboside, respectively, is also described.     

Detection and identification of cytokinins produced by mycorrhizal fungi

Several fungi including six species of the genus Rhizopogon, 22 species of Hebeloma and one of Agaricus have been screened for production of cytokinins. The screening was done by culturing cytokinin-requiring soybean callus tissue alongside the fungus on a medium lacking a cytokinin supply. Growth of the soybean callus indicated production of cytokinins by the fungus. Of the fungi tested, only R. ochraceorubens A. H. Smith gave off sufficient cytokinin to be detected. Although a number of mycorrhizal species are now known to make and give off cytokinins, an even larger number apparently do not do so under the conditions of screening employed. An unidentified ectendotrophic species definitely gave off trans-zeatin, which has been crystallized, and probably trans-ribosylzeatin. Suillus punctipes (Pk.) Sing. apparently produced the same two cytokinins.     

A Cytokinin-binding Protein from Wheat Germ: Isolation by Affinity Chromatography and Properties

A cytokinin-binding protein has been isolated from wheat germ via ammonium sulfate precipitation, carboxymethyl Sephadex chromatography, and affinity chromatography on a column substituted with a derivative of kinetin riboside. On Sephadex G-200, the protein migrated with an apparent molecular weight of 122,000 daltons. The dissociation constant for kinetin was determined by equilibrium dialysis to be 1.2 micromolar; N⁶-benzylaminopurine and N⁶-(Δ²-isopentenyl)adenine were also strongly bound. Little affinity was exhibited toward either cis-zeatin or trans-zeatin.     

Cytokinin biosynthesis in crown gall tissue of Vinca rosea: Metabolism of isopentenyladenine

When isopentenyl[8-¹⁴C]adenine was incubated with crown gall tumour tissue of Vinca rosea, it was stereospecifically hydroxylated to trans-zeatin and its derivatives, which are the endogenous free cytokinins in this tissue. Adenine, adenosine and adenine nucleotides were the major degradation products.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.