Plant growth regulators as putative physiological markers of developmental stage in Prunus persica

Prunus persica plants of different ages and statesof maturation were analysed to compare their phytohormonal status. Endogenouslevels of indole-3-acetic acid (IAA), abscisic acid (ABA), and severalcytokinins (Cks): zeatin (Z), zeatin riboside ([9R]Z), dihydrozeatin ((diH)Z),dihydrozeatin riboside ((diH)Z[9R]), isopentenyl adenine (iP) and isopentenyladenosine ([9R]iP), were measured in order to determine their possible use asphysiological indices of phase change and maturation. A decrease in Ck levels(Z, [9R]Z, (diH)Z, (diH)[9R]Z, and iP, [9RiP) was found from the embryonic tojuvenile stage as well as a decrease in the ratio of iP-type (iP and[9R]iP)/Z-type Cks paralleling the increase in tree age. ABA levels increasedduring maturation in Prunus persica and the ratio ofCks/IAA decreased with tree age. From our results, we propose that the balancesof Cks/IAA and iP-type/Z-type Cks are good indices of different developmentalstates in Prunus persica.     

Chicken egg yolk antibodies for cytokinin analysis

The production, isolation, and purification of specific chicken immunoglobulins (Igs) against three main groups of naturally occurring cytokinins are reported. The specific Igs directed against, respectively, zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine are extracted from the egg yolk and used in radioimmunoassays that allow the quantification in parallel of pmol of the cytokinins in plant extracts. As little as 50 fmol of zeatin riboside, 20 fmol of isopentenyladenosine, and 40 fmol of dihydrozeatin riboside can be detected. The levels of cytokinins measured in the radio-immunoassay correlate well with physicochemical analysis methods such as high performance liquid chromatography (HPLC) with UV spectrum detection and HPLC-coupled mass spectrometric detection. Cross-reactivity studies indicate that the assay is not affected by most of the structurally related compounds. The respective antibody preparations recognized zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine and the corresponding free bases. The results obtained when analyzing crude plant extracts are expressed as zeatin riboside equivalents, dihydrozeatin riboside equivalents, and isopentenyladenosine equivalents.Abbreviations B binding activity - B ₀ maximal binding - B ₁ unspecific binding - GC gas chromatography - HPLC high performance liquid chromatography - LC-MS HPLC-coupled mass spectrometry - MOPS 4-morpholinepropanesulfonic acid - RIA radioimmunoassay - TBS Tris-buffered saline - (diH)Z dihydrozeatin - (diH) [9R]Z dihydrozeatin riboside - iP isopentenyladenine - [9R]iP isopentenyladenosine - Z zeatin - [9R]Z zeatin riboside - [9G]iP isopentenyladenine-9-glucoside - [9R-5P]iP isopentenyladenosine-5-monophosphate     

Solid-phase extraction of cytokinins from aqueous solutions with C18 cartridges and their use in a rapid purification procedure

Eleven cytokinins-including bases, ribosides, glucosides, and ribotides-were tested for their retention on C₁₈ cartridges that were washed with 40 mL of water or a dilute acid at pH 3. Cytokinins were then eluted with methanol and analyzed by high performance liquid chromatography (HPLC). All pure cytokinin were well retained when the cartridge was washed with water, but Z and (diH)Z were less well retained at pH 3. The ribotides required 80% methanol for elution. Cotton leaf tissue (500 mg dry wt) was spiked with cytokinins, extracted with 80% methanol, and the extract bulk purified with hexane, insoluble polyvinylpyrrolidone, and minicolumns (strong anion exchange, amino, and C₁₈ cartridges). Ribotides, added to leaf tissue, could not be recovered as ribotides; it was necessary to hydrolyze and purify them as ribosides. The cytokinins were separated and analyzed by HPLC on strong cation exchange and C₁₈ columns. Recoveries through the entire procedure averaged 70%.Cytokinin abbreviations (diH)Z Dihydrozeatin - (diH)Z dihydrozeatin riboside - (diH)[9R]Z trans-zeatin - Z t-zeatin riboside - [9R]Z t-zeatin-O-glucoside - (OG)Z t-zeatin riboside-O-glucoside - (OG)[9R]Z t-zeatin riboside-5-monophosphate - [9R-5P]Z N6(^2-isopentenyl)adenine - iP N6(^2-isopentenyl)adenosine - [9R]iP N6(^2-isopentenyl)adenosine-5-monophosphate-[9R-5P]iP     

Identification of the cytokinins in red pine seedlings

Zeatin-9-riboside was identified in shoots and roots of Pinus resinosa by GC-MS analysis of its permethyl derivative. Based on their chromatographic properties on Sephadex LH-20 and C₁₈ HPLC, and their susceptibility to enzymatic degradation, several other cytokinins have been tentatively identified. The basic fraction of both the roots and shoots contained zeatin, whereas the shoots contained dihydrozeatin-O-glucoside and the roots contained zeatin-O-glucoside. Zeatin-9-riboside monophosphate, isopentenyladenosine monophosphate ([9R-5P]iP) and glucosyl phosphate derivatives were detected in the acidic fractions from both roots and shoots. There were equivalent amounts of [9R-5P]iP in both roots and shoots. The presence of equivalent amounts of [9R-5P]iP in both the roots and shots suggests that cytokinin biosynthesis may be occurring in both locations.Abbreviations AMP adenosine-5-monophosphate - BAP benzylaminopurine - BSA bovine serum albumin - BuOH butan-1-ol - CK cytokinin - (diH)Z dihydrozeatin - (diH OG)Z dihydrozeatin-O-glucoside - (diH OG)[9R]Z dihydrozeatin-9-riboside-O-glucoside - DW dry weight - EtOH ethanol - FW fresh weight - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - [9R]iP isopentenyladenosine - [9R-5P]iP isopentenyladenosine monophosphate - MeOH methanol - PVP polyvinylpyrrolidone - RFE rotary film evaporation - TEAB triethyl ammonium bicarbonate - Z zeatin - [9R]Z zeatin-9-riboside - (OG)Z zeatin-O-glucoside - [7G]Z zeatin-7-glucoside - [9R-5P]Z zeatin-9-riboside monophosphate     

Endogenous cytokinins in vegetative shoots of peas

In G2 peas senescence only takes place in long days. In order to determine the role of cytokinins in this process the endogenous cytokinins from vegetative shoots of G2 peas were characterized using gas chromatography-mass spectroscopy following purification by HPLC. Cytokinins were extracted and purified with and without the addition of ¹⁵N labelled internal standards of several cytokinins to estimate cytokin content by isotope dilution in the mass spectra. Samples without internal standards were bioassayed after HPLC. Bioassays showed the presence of zeatin, zeatin riboside and zeatin-0-glucoside. The presence of zeatin was confirmed by its mass spectrum of its permethylated derivative. Tentative identification of zeatin riboside, zeatin-0-glucoside, dihydrozeatin, and dihydrozeatin-0-glucoside was obtained by the coincidence of the major ion for the permethylated natural and ¹⁵N labelled internal standards on GC-MS, and the similar coincidence of ions for permethylated zeatin riboside-0-glucoside by direct probe MS. There was no indication of the presence of significant quantities of zeatin-7-glucoside or zeatin-9-glucoside. The amounts in the tissue ranged from 200–1000 ng/kg fresh weight for each cytokinin and about 2–4 g/kg fresh weight for total cytokinins. There was no apparent difference in the levels in mature but pre-senescent shoots grown in long days and short days indicating that apical senesecence in G2 peas does not appear to be induced by a decline in cytokinin level in the shoots.Cytokinin abbreviations CK Cytokinin - Z trans zeatin - [9R]Z t-zeatin riboside - [9R-5P] Z t-zeatin riboside-5-monophosphate - (OG)Z t-zeatin-0-glucoside - (OG)[9R]Z t-zeatin riboside-0-glucoside - [7Z]G t-zeatin-7-glucoside - [9G]Z t-zeatin-9-glucoside - (diH)Z dihydrozeatin - (diH)[9R]Z dihydrozeatin riboside - iP N6(²-isopentenyl) adenine - [9R]iP N6(²-isopentenyl) adenosine Work performed while PJD was on leave at the University College of Wales at Aberystwyth.     

Hormones and Pod Development in Oilseed Rape (Brassica napus)

The endogenous levels of several plant growth substances (indole acetic acid, IAA; abscisic acid, ABA; zeatin, Z; zeatin riboside, [9R]Z; isopentenyladenine, iP; and isopentenyladenosine, [9R]iP were measured during pod development of field grown oilseed Rape (Brassica napus L. var oleifera cv Bienvenu) with high performance liquid chromatography and immunoenzymic (enzyme-linked immunosorbent assay, ELISA) techniques. Results show that pod development is characterized by high levels of Z and [9R]Z in 3 day old fruits and of IAA on the fourth day. During pod maturation, initially a significant increase of IAA and cytokinins was observed, followed by a progressive rise of ABA levels and a concomitant decline of IAA and cytokinin (except iP) levels. The relationship between hormone levels and development, especially pod number, seed number per pod, and seed weight determination, will be discussed.     

An evaluation of the role of cytokinins in the development of abnormal inflorescences in oil palms (Elaeis guineensis Jacq.) regenerated from tissue culture

Tissue cultures and regenerant plants from cell lines producing palms with normal and abnormal flowers were analyzed for cytokinin content and compared with zygotic embryos and seedlings. Immature inflorescences at the critical stage of flower development dissected from normal and abnormal palms were also analyzed. High performance liquid chromatography (HPLC)/radioimmunoassay and HPLC/enzyme-linked immunosorbent assay methods were used over a period of several years to measure the isoprenoid cytokinins. The results of analyses of endogenous aromatic cytokinins, present at comparable levels, will be reported separately. Oil palm cultures and regenerant plants contained relatively high concentrations of the 9-glucosides of isopentenyladenine ([9G]iP) and zeatin ([9G]Z). The predominant biologically active isoprenoid cytokinin present was zeatin riboside ([9R]Z), with lesser amounts of isopentenyladenine (iP) and isopentenyladenosine ([9R]iP). There was evidence of small amounts of dihydrozeatin compounds, but high concentrations (mainly as dihydrozeatin-9-glucoside ([9G]DHZ)) were confined to the haustorium of the zygotic embryo. Callus tissue contained very low concentrations of cytokinin. Frequently only [9G]iP could be detected, at about 1 pmol · g^-1 fresh weight, with [9R]Z at less than 0.05 pmol · g^-1. In comparison, nodular embryogenic tissues in vitro contained between 30 and 1,500 pmol · g^-1 of [9G]iP, 5–50 pmol · g^-1 of [9G]Z, and up to 12 pmol · g^-1 of [9R]Z. Shoots of regenerant plantlets and seedlings contained lower concentrations of [9G]iP (3–30 pmol · g^-1), although this was still the predominant cytokinin. [9R]Z and [9G]Z were present at between 2 and 15 pmol · g^-1, with iP at 1–5 pmol · g^-1 and [9R]iP at between 1 and 12 pmol · g^-1. Seedlings contained similar amounts with the exception of a lower [9G]iP content (5–10 pmol · g^-1) and more [9R]iP (10–20 pmol · g^-1). Root tissues of ramets contained significantly higher concentrations of [9G]iP than shoots. Comparison of two isogenic lines of one clone giving rise to normal and abnormal palms showed significantly higher concentrations of [9R]Z and [9G]Z in the normal than in the abnormal line and, in embryoids only, higher [9G]iP in the normal line. In all other cases the between-done differences were greater than any normal/abnormal differences. There was a general tendency for increased concentrations of [9G]iP in abnormal lines and for this compound to be in a higher concentration in embryoids and plants derived from culture than in zygotic embryos and seedlings. Analysis of cytokinins in immature female inflorescences of normal and abnormal palms of a single clone showed the abnormal inflorescences to have higher concentrations of [9R]Z and [9R]DHZ and less [9G]Z than the normal inflorescences at comparable stages of development.Abbreviations HPLC high performance liquid chromatography - [9G]iP 9-glucoside of isopentenyladenine - [9G]Z 9-glucoside of zeatin - [9R]Z zeatin riboside - iP isopentenyladenine - [9R]iP isopentenyladenosine - [9G]DHZ dihydrozeation-9-glucoside - ELISA enzyme-linked radioimmunosorbentassay - ANOVAR analysis of variance     

Changes in the Chlorophyll Contents of Leaves and in Levels of Cytokinins in Root Exudates during Ripening of Rice Cultivars Nipponbare and Akenohoshi

Changes in fluxes of cytokinins in exudates transported viathe xylem from roots of rice plants cvs. Nipponbare (a standardJapanese cultivar) and Akenohoshi (a slowly senescing cultivar)were measured by mass spectrometry with deuterium-labeled standards.The fluxes of zeatin (Z), trans-ribosylzeatin (trans-RZ), N⁶-isopentenyladenine(iP), and "conjugated Z" (Z in the hydrolysates of highly polarfractions) decreased from heading to the late ripening stagein both cultivars. At the late ripening stage, iP and Z couldno longer be detected, while the flux of N⁶-isopentenyladenosine(iPA) increased slightly. In Akenohoshi, conjugated Z was thepredominant cytokinin from heading to the middle of the ripeningstage. The flux of each of the cytokinins in Akenohoshi washigher than that in Nipponbare at every time point, with theexception of the flux of iPA just after heading. The total concentrationof cytokinins in the xylem exudate of Akenohoshi was higherthan that of Nipponbare after the middle of ripening stage.The chlorophyll content of the third leaves, which were senescingrapidly, was significantly correlated with the flux of totalcytokinins per plant or per unit leaf area. These results suggestthat the larger amounts of cytokinins, in particular conjugatedZ, transported from the roots to the shoots caused the slowsenescence of leaves in Akenohoshi during the ripening stage. (Received May 9, 1994; Accepted July 1, 1995)     

Turnover of O-Glucosides of Dihydrozeatin and Dihydrozeatin-9-riboside During the Cell Growth Cycle of Photoautotrophic Cell Suspension Cultures of Chenopodium rubrum

The cellular levels of O-glucosides of ³H-(diH)Z and ³H-(diH)[9R]Z, the major short-term metabolites of ³H-(diH)Z having been exogenously supplied to photoautotrophically growing suspension cell cultures of Chenopodium rubrum, decreased significantly during further culture, irrespective of whether the cells were maintained in the stationary phase or were transferred to conditions restoring cell divison. Metabolism of both compounds was more pronounced during the active growth phase than during the stationary phase. The O-glucosides were converted preferentially to polar compounds of as yet unknown nature, which were partly excreted into the medium. The cellular pools of both glycosides remained compartmented within the vacuole. In contrast to the O-glycosides, the small cellular pools of the aglycones ³H-(diH)Z and ³H-(diH)[9R]Z maintained their level during the experimental period of 30 days. Small amounts of the glucosides, as well as of the aglycones, were recovered from the medium and could have resulted from the lysis of a few cells. The results demonstrate, for the first time, that O-glucosides of cytokinins are not irreversibly deposited within the vacuole of plant cells but may serve to maintain a small, but more or less constant pool of extra-vacuolar, presumably cytosolic, aglycones. (DiH)Z and its derivatives could be demonstrated to be endogenous cytokinins of Chenopodium rubrum suspension cultured cells occurring along with those of the isopentenyladenine and zeatin types.     

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures

Summary Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.Abbreviations BM basal medium - BA 6-benzyladenine - CIM callus induction medium - CPM callus proliferation medium - (diH)Z dihydrozeatin - DW dry weight - ELISA enzyme linked immunoassay - FW fresh weight - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - NAA 1-naphthaleneacetic acid - PBS phosphate buffer - PGR plant growth regulator - (diH)[9R]Z 9--D-ribofuranosyl-(diH) - [9R]iP 9--Dribofuranosyl-iP - [9R]Z 9--D-ribofuranosylzeatin - TBS trishydroxymethyl-aminomethane buffer - Z zeatin     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Immunocytochemical location of endogenous cytokinins in buds of kiwifruit (Actinidia deliciosa) during the first hours of in vitro culture

Post-embedding immunocytochemical techniques using peroxidase–antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9--D-ribofuranosil-N ⁶-(²-isopentenil) adenina ([9R]iP), 9--D-ribofuranosyl-zeatin ([9R]Z) and 9--D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72h in liquid medium. Subcellular immuno-localization was performed in cells from explants grown for 35d on agar solidified-medium and for 30min, 4 and 16h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4h of culture in the cytoplasm, and at 16h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.     

Benzyladenine and derivatives-their significance and interconversion in plants

Recently benzyladenine has been isolated as a natural cytokinin from a number of plants. The natural occurrence of this cytokinin will change the attitude with which physiologists view this hormone. This review attempts to put into context what is known about this cytokinin and its derivatives and to compare and contrast its metabolism and the function and physiological action of its various metabolites. Nothing is known about the biosynthesis of benzyladenine. Its structure would suggest that its biosynthetic pathway may differ considerably from that of zeatin and iso-pentenyladenine.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - [9R]BA BA ribonucleoside - [9R-MP]BA BA nucleotide - [9R-DP]BA BA dinucleotide - [9R-TP]BA BA trinucleotide - [3G]BA BA 3 glucoside - [7G]BA BA 7 glucoside - [9G]BA BA 9 glucoside - [9R-G]BA BA 9-ribosylglucoside - [9Ala]BA BA alanine-conjugate - (2OH)BA BA ortho-OH - (2OH)[9R]BA BA ortho-Oh-riboside - KN kinetin - [9R]KN KN ribonucleoside - DHZ dihydrozeatin - Z trans-zeatin - [9R]Z zeatin ribonucleoside - [7G]Z zeatin-7-glucoside - [9G]Z zeatin-9-glucoside - [9Ala]Z zeatin alanine-conjugate - (OG)[9R]Z O-glucoside of zeatin ribonucleoside - [9R-MP]Z zeatin nucleotide - iP iso-pentenyladenine - [9R]iP iP ribonucleoside     

A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

Summary Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 × g particulate fraction. Binding of [8-¹⁴C]-benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at pH 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (K_d) of 33 ± 6 nM. The number of binding sites found was 1,100 ± 120 fmol g^–1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [9 R] Z, [9 R] A.     

Thermoperiodic effect on flowering and endogenous hormonal status in Dendrobium (Orchidaceae)

Two year-old cloned plants of Dendrobium Second Love were submitted to 25 °C (light) and 10 °C (dark) under a 12 h photoperiod (60 μmol m⁻¹ s⁻¹) for 30 days. The endogenous levels of IAA, ABA, and the cytokinins Z, [9R]Z, iP, and [9R]iP were measured 15, 22, and 30 days after the start of the thermoperiodic treatment in lateral buds and leaves. The endogenous levels of IAA and cytokinins, especially the zeatin-derived forms, increased significantly in buds after 15 days of treatment. On the other hand, the amount of ABA decreased progressively and significantly throughout the treatment. The treatment conspicuously accelerated flower-bud development. The found correlation suggests that hormones are involved in the signal transduction pathway of thermoperiodic flowering control.     

Endogenous plant growth regulators in carnation tissue cultures under different conditions of ventilation

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins (zeatin, zeatin riboside, dihydrozeatin, (diH)Z, dihydrozeatin riboside, (diH)[9R]Z, N⁶-isopentenyl adenine and N⁶-isopentenil adenine riboside) levels were evaluated in normal (N) and hyperhydric (H) microplants of Dianthus caryophyllus cultured under different aeration conditions in hormone-free liquid medium. The morphological differences between N and H explants grown under ventilated conditions were correlated with differences in their endogenous hormonal levels: after 15 and 30 days of culture, H explants showed lower IAA and ABA contents than N explants, as well as higher cytokinin levels, mainly of (diH)Z and (diH)[9R]Z. This was associated with less tissue differentiation and with an inability of H microplants to survive under ex vitro conditions. However, these relationships could not be observed between H and N explants grown under non-ventilated conditions probably due to the difficulty in discerning the plant status (N or H) and therefore, an underestimation of H microplants. This assumption is supported by the low ability for acclimatization to ex vitro of N plants grown without ventilation.     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N⁶-(²-isopentenyl)adenine ([2,8-³H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent K_m value of the enzyme was 4.3 M for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-³H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl₂) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent K_m value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.Abbreviations Ade adenine - iP N⁶-(²-isopentenyl)adenine - [2,8-³H]iP [2,8-³H]-N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - (diH)iP N⁶-isopentyladenine - (diH)Z dihydrozeatin - BAP N⁶-benzyladenine - ( _o OH)[9R]BAP N⁶-(o-hydroxybenzyl)adenosine - (mOH)[9R]BAP N⁶-(m-hydroxybenzyl)adenosine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthalene-1-acetic acid - ABA abscisic acid - Con A concanavalin A     

Effects of NO3−, NH4+ and urea nutrition on endogenous levels of IAA and four cytokinins in two epiphytic bromeliads

The long-term effects of different nitrogen sources on the endogenous IAA and cytokinin levels in two bromeliad species were investigated. In nature, Vriesea philippocoburgii is a tank-forming epiphytic bromeliad which uses the tank water reservoir as a substitute for soil, whereas Tillandsia pohliana is a tankless atmospheric epiphytic species. A culture was established from seeds germinated in aseptic condictions, and the plantlets were grown for 6 months in a modified Knudson medium to which was added 8 mol m⁻³ of nitrogen in the form of NO₃⁻, NH₄⁺ or urea. The hormonal contents of the bromeliad shoots were determined by means of high-performance liquid chromatography (HPLC), coupled to an enzyme-linked immunosorbent assay (ELISA) for indole-3-acetic acid (IAA), isopentenyladenine (iP), isopentenyladenosine ([9R]iP), zeatin (Z) and zeatin riboside ([9R]Z). Nitrogen supplied in the form of urea gave the highest values of fresh and dry weights for both species, and this was positively correlated to IAA levels. The cytokinin patterns showed that isopentenyladenosine was the predominant form for both species in all samples. However, urea induced the highest level of this riboside form and also the highest level of total cytokinins for V. philippocoburgii, while NH₄⁺ had the same effect on the atmospheric species. These results are discussed in terms of the different growth habits of these two species in nature. It is suggested that urea may be an important source of nitrogen often found inside the tank of V. philippocoburgii. NO₃⁻ treatment increased the IAA/Cks balance, mainly for V. philippocoburgii, while urea and NH₄⁺ shifted this ratio in favour of cytokinins, thus apparently inhibiting root development in both species.     

Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum

[³H]Dihydrozeatin supplied to photoautotrophically growing cell suspension cultures of Chenopodium rubrum was rapidly taken up and metabolized by the cells. The predominant metabolites in extracts of the cells were [³H]dihydrozeatin-O-glucoside and [³H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [³H]dihydrozeatin and [³H]dihydrozeatin riboside, which were both present to a minor extent in cell extracts, were both present to a minor extent in cell extracts, were localized predominantly outside the vacuole. Analysis of the culture medium at the end of the 36-h incubation period showed that there had been an efflux of [³H]dihydrozeatin metabolites out of the cells. Whereas [³H]dihydrozeatin riboside was found to be the major extracellular [³H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [³H]dihydrozeatin could be detected in the medium. The differential compartmentation of [³H]dihydrozeatin metabolites found with the C. rubrum suspension-culture system is discussed with respect to possible mechanisms governing the metabolism of cytokinins in plants cells.Abbreviations (diH)Z dihydrozeatin - (diH) [9R]Z 9--D-ribofuranosyl dihydrozeatin - HPLC high-performance liquid chromatography - ODS octododecyl silica - PEP phosphoenolyruvate