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.     

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−³H]trans-zeatin ([2−³H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−³H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−³H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Cytokinins in cut carnation flowers: I. The complex in ovaries

Tentative identification of the cytokinins present in extracts of Dianthus caryophyllus ovaries using High Performance Liquid Chromatography and Radioimmunoassay techniques, revealed the presence of trans-ribosylzeatin, trans-zeatin, dihydrozeatin and N⁶ (²-isopentenyl)adenine. In addition slow moving compounds (paper chromatography) which could be hydrolysed by -glucosidase were also detected. After hydrolysis the active compounds co-chromatographed with zeatin and ribosylzeatin.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

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     

Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells

Summary Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP dimethylallylpyrophosphate - iP isopentenyladenine - iPA isopentenyladenosine - iPMP isopentenyladenosine 5-monophosphate - IPT isopentenyltransferase - trans-Z trans-zeatin     

Effects of trans- and cis-zeatin and optical isomers of synthetic cytokinins on protein kinase activity in vitro

The effects of cis- and trans-zeatin on the activity of the protein kinase associated with barley leaf chromatin were studied. Substances tested were added directly into the incubation medium for enzyme activity estimation. Only trans-zeatin activated the chromatin-associated protein kinase. Maximum activation was detected at a trans-zeatin concentration of 10^–9 m. cis-Zeatin had no activity in a range of concentrations from 10^–10 to 10^–5 m. Comparison of the R-(-)-N ⁶-1-(1-naphthyl)ethyl-1H-purine-6-amine (R-NEPA) and S-(+)-N ⁶-(1-naphthyl)ethyl-1H-purine-6-amine (S-NEPA) effects on the enzyme activity showed that only S-NEPA activated the protein kinase from barley leaf chromatin, whereas R-NEPA had no such effect. The data on the effect of other synthetic analogues of cytokinin on the protein kinase activity are also presented. The results are discussed in terms of the specificity and sensitivity of the in vitro response of the chromatin-associated protein kinase from barley leaves to cytokinins. The advantages and limitations of this in vitro assay to test cytokinin activity are also considered.Abbreviations BAP N ⁶-benzylaminopurine - R-NEPA R(-)-N ⁶-(1-naphthyl)ethyl-1H-purine-6-amine - S-NEPA S-(+)-N ⁶-1-(1-naphthyl)ethyl-1H-purine-6-amine - CBP cytokinin-binding protein     

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.     

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.     

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.     

Micropropagation of globe artichoke (Cynara scolymus L.) from underground dormant buds (“ovoli”)

Summary Side shoots excised from underground dormant buds ofCynara scolymus L. were used as primary explants to establishin vitro cultures. A 3×3 factorial experiment with all possible combinations of three concentrations (0.5, 1.0, 2.0 mg/liter or 2.22, 4.44, 8.88 μM) ofN ⁶-benzyladenine (BA) and three concentrations (0, 0.1, 0.2 mg/liter or 0, 0.54, 1.07 μM) of 1-naphthaleneacetic acid (NAA) was used to determine the optimum growth regulator combination for shoot multiplication. The highest rate of axillary shoots was induced on Murashige and Skoog agar medium supplemented with 0 mg NAA/liter and 1.0 mg BA/liter (4.44 μM). Other cytokinins tested (kinetin, zeatin, and 2-isopentenyl-adenine were less effective than BA in inducing axillary shoot growth. Up to 60% of elongated microshoots rooted after 5 weeks on 1/2 MS agar medium supplemented with 2 mg/liter (11.42 μM) indole-3-acetic acid (IAA). Seventy percent of rooted plantlets were transferred successfully into soil. Plants are under evaluation for their genetic uniformity and clonal fidelity.     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

Cytokinins: synthesis and biological activity of geometric and position isomers of zeatin

Geometric and position isomers of zeatin and of ribosylzeatin and other compounds closely related to zeatin have been tested in the tobacco (Nicotiana tabacum var. Wisconsin No. 38) bioassay. None was more active than zeatin itself. There was a much greater difference in activity (> 50-fold) between trans- and cis-zeatin than between trans-isozeatin [6-(4-hydroxy-2-methyl-trans-2-butenylamino) purine] and cis-isozeatin [6-(4-hydroxy-2-methyl-cis-2-butenylamino) purine], the latter being less active than cis-zeatin and trans-isozeatin. Higher concentrations were required for equivalent callus growth stimulated by the 9-ribosyl derivatives, which followed an order of decreasing activity: ribosyl-trans-zeatin > ribosyl-cis-zeatin > ribosyl-trans-isozeatin > ribosyl-cis-isozeatin, corresponding roughly to that of the bases. The effect of side chain, double bond saturation was to diminish the activity, and in the dihydro series the shift of the methyl group from the 3- to the 2-position in going from dihydrozeatin to dihydroisozeatin [6-(4-hydroxy-2-methylbutylamino) purine] resulted in a 70-fold decrease in activity. cis-Norzeatin [6-(4-hydroxy-cis-2-butenylamino) purine], which was less than one-fifth as active as cis-zeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3,6-dihydro-1,2-oxazin-2-yl) purine] was about 1/100 as active as cis-norzeatin. These findings delineate completely the effect on the cytokinin activity of zeatin of variation in side chain geometry, presence and position of the methyl substituent, presence and geometry of hydroxyl substitution, presence of the double bond, and of side chain cyclization.     

Dynamics of production oftrans-zeatin andtrans-zeatin riboside by immobilized cytokinin-autonomous and cytokinin-dependent tobacco cells

Two strains of cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) differing in their requirement for exogenous cytokinins (cytokinin-dependent and cytokinin-autonomous) were immobilized on polyphenylenoxide (Sorfix) activated with glutaraldehyde. Columns packed with immobilized cells were continually eluted with diluted Murashige and Skoog's medium lacking or supplemented with synthetic cytokinin (6-benzylaminopurine; BA). Purified samples of column eluates were fractionated by HPLC, andtrans-zeatin (t-Z) andtrans-zeatin riboside (t-ZR) content was estimated by enzyme immunoassay. Both cytokinin-autonomous and cytokinin-dependent tobacco cells produced and excretedt-Z and its riboside, and there were significant quantitative differences between the strains. The steady-state excretion rate oft-Z was 19.8 ng · g^–1 dw · h^–1 and 4 ng · g^–1 dw · h^–1, respectively, and that oft-ZR 4 ng · g^–1 dw · h^–1 and 1 ng · g^–1 dw · h^–1, respectively. Exposure of cytokinin-dependent cells to BA after 72 h of starving for this synthetic cytokinin caused temporary increase in excretion of both zeatin and its riboside. After the application of 5 M BA for 24 h, the excretion rate oft-ZR reached 5 ng · g^–1 dw · h^–1 (5-fold increase), and that oft-Z achieved 12 ng · g^–1 dw · h^–1 (3-fold increase). The elevation oft-Z excretion was delayed about 13 h compared witht-ZR excretion, which started increasing almost immediately after BA application. A pulse of BA in lower concentration (1.5 M for 30 h) provoked lower response.