The synthesis and biological properties of 8-azido-N 6-benzyladenine,a potential photoaffinity reagent for cytokinin

A cytokinin photoaffinity reagent, 8-azido-N ⁶-benzyladenine (8N₃BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N–1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N₃BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N₃BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.Abbreviations BA N ⁶-benzyladenine - 8N₃BA 8-azido-N ⁶-benzyladenine - PMR proton magnetic resonance - TLC thin-layer chromatography - UV ultraviolet In partial fulfillment of requirements for the Ph.D. degree at Michigan State University     

Chemical inhibition of cell wall formation and cytokinesis,but not of nuclear division,in protoplasts of Nicotiana tabacum L. cultivated in vitro

The effect of cytochalasin B, colchicine, coumarin and 2,6-dichlorobenzonitrile on cell wall formation and cellular division was studied by light and electron microscopy with tobacco mesophyll protoplasts cultivated in vitro. 2,6-dichlorobenzonitrile was found to be the most effective and reversible inhibitor of cell wall formation. The other inhibitors caused irreversible damage and/or inhibited mitosis. In protoplasts cultivated in the presence of 2,6-dichlorobenzonitrile the total inhibition of cell wall formation had no effect on nuclear division, but cytokinesis was totally inhibited so that multinucleate protoplasts were obtained.Abbreviations DB 2,6-dichlorobenzonitrile=dichlobenil - CB cytochalasin B     

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex

Summary Tobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201–212, 1984).Poly(A)⁺RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)⁺RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells.Dot-blot and northern-blot hybridizations of poly(A)⁺RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304–7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)⁺RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)⁺RNA.Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613     

Effect of exogenous cytokinins,auxins and adenine on cytokinin <Emphasis Type="Italic">N</Emphasis>-glucosylation and cytokinin oxidase/dehydrogenase activity in de-rooted radish seedlings

The role of cytokinin N-glucosylation and degradation by cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) in response to application of exogenous auxins (2,4-dichlorophenoxyacetic acid [2,4-D] and -naphthaleneacetic acid [NAA]) and cytokinins (N ⁶-benzyladenine [BA] and trans-zeatin [Z]) was investigated in de-rooted seedlings of Raphanus sativus L. cv. Rampouch. Both auxins applied for 24 h at 1 and 10 M concentration increased N-glucosylation of exogenously applied [³H]dihydrozeatin (DHZ) by up to 20%. The level of endogenous 7N-glucosides (of Z, isopentenyladenine [iP] and DHZ) was increased by 2,4-D and NAA at 10 M concentration by 28 and 23%, respectively, the level of Z being decreased by 90 and 59%, respectively. 2,4-D and NAA suppressed CKX activity ca. by half. Exogenous cytokinins Z and BA applied at 1 and 10 M concentration stimulated 7N-glucosylation of [³H]DHZ (by up to 40%). BA both at 1 and 10 M, increased the level of endogenous Z by up to 35% and that of 7N-glucosides by up to 27%. BA application also strongly stimulated CKX activity (by up to 180%). Feeding with 1 and 10 M Z resulted in ca. 100-fold and 2000-fold increase of Z level, respectively. The main metabolite, Z7G, was increased ca. 6-fold and 60-fold, respectively. Levels of Z 9-glucoside (Z9G), trans-zeatin riboside (ZR) and Z O-glucoside (ZOG) were elevated to lesser extent. As compared to BA, Z had only negligible effect on CKX activity. Adenine (1–500 M) was preferentially 7N-glucosylated inhibiting competitively 7N-glucosylation of [³H]DHZ. At high concentrations (100–500 M) it increased endogenous levels of active cytokinins, especially of Z, however, it had no effect on CKX activity. Cytokinin N-glucosylation proved to be involved in down-regulation of active cytokinins in response to auxin and in the re-establishment of cytokinin homeostasis following application of exogenous cytokinins.     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type     

Time course of hormonal control of the first mitosis in tobacco mesophyll protoplasts cultivated in vitro

The presence of auxin and cytokinin is necessary for the induction of mitosis in tobacco mesophyll protoplasts cultivated in vitro. In their absence, protoplasts firstly accumulate inhibitors of mitosis in the culture medium, possibly because of non-coordinated cell-wall synthesis, and secondly evolve a nonmitotic and degenerative metabolism. By changing the intoxified medium, it is possible to show that auxin is necessary from the beginning of culture, while cytokinin is only required later to allow a step in the development of the mitotic apparatus.Abbreviations 2.4-D 2.4 dichlorophenoxyacetic acid - 6-BA 6-benzyladenine - NAA naphthaleneacetic acid - IAA indoleacetic acid     

Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea

cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The trans-isomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - TMSi trimethylsilyl - tRNA transfer RNA - ZR zeatin riboside     

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. Havana 425 becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10^–3 per cell generation, was 100–1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10^–10 M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.     

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.     

Regulators of cell division in plant tissues

The cytokinins in certain fractions prepared from extracts of immature sweet-corn (Zea mays L.) kernels using polystyrene ion-exchange resins have been further investigated. Cytokinins active in the radish cotyledon bioassay were purified from these fractions and identified as 9--D-glucopyranosylzeatin, 9--D-glucopyranosyldihydrozeatin, O--D-glucopyranosylzeatin. and O--D-glucopyranosyl-9--D-ribofuranosylzeatin. In addition, compounds which resemble zeatin and its glycosides in chromatographic behaviour and in ultraviolet absorption characteristics were purified from extracts of the same material by high-performance liquid chromatography. In addition to zeatin and zeatin riboside, the following compounds were identified unambiguously: O--D-glucopyranosyl-9--D-ribofuranosyldihydrozeatin, O--D-glucopyranosyldihydrozeatin, and hihydrozeatin riboside. A further compound was tentatively identified as O--D-glucopyranosylzeatin, and at least two unidentified compounds appeared to be new derivatives of zeatin. In identifying the above compounds, chemical-ionization mass spectrometry proved to be an invaluable complementary technique, yielding spectra showing intense protonated-molecular-ion peaks and also prominent structure-related fragmentation that was either not evident or very minor in the electron-impact spectra. An assessment of the relative importance of the various possible mechanisms for cytokinin modification and inactivation in mature sweet-corn kernels was made by supplying [³H]zeatin and [³H]zeatin riboside to such kernels after excision. The principal metabolites of zeatin were adenine nucleotides, adenosine and adenine, while little of the metabolite radioactivity was attributable to known O-glucosides. Adenine nucleotides and adenine were the principal metabolites of zeatin riboside, while lesser metabolites were identified as adenosine, dihydrozeatin, and the O-glucosides of dihydrozeatin and dihydrozeatin riboside. Side-chain cleavage, rather than side-chain modification, appears to be the dominant form of cytokinin metabolism in mature sweet-corn kernels.Abbreviations CI-MS chemical-ionization mass spectrum - EIMS electron-impact mass spectrum - GC-MS combined gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - M⁺ molecular ion - MH⁺ protonated molecular ion - TLC thin-layer chromatography - TMS trimethylsilyl - UV ultraviolet XXVII=Letham et al. (1979)     

Cytokinins promotion of flowering in Cymbidium ensifolium var. misericors in vitro

Callus-derived rhizomes of Cymbidium ensifolium var. misericors produced flowers precociously on a defined basal medium (1/2MS) containing of NAA with thidiazuron (TDZ), N⁶-(2-isopentenyl) adenine (2iP) or N⁶-benzyladenine (BA) within 100 d of culturing. Among eight cytokinins tested, TDZ at 3.3–10 µM or 2iP at 10–33 µM combined with 1.5 µM NAA were the most effective combinations for achieving flower induction in vitro. These undersized flowers were physically normal and bloomed for two weeks in vitro.     

The frequency of embryogenic pollen grains is not increased by in vitro anther culture in Nicotiana tabacum L.

The numbers of embryogenic (S) grains present in in-situ mature anthers of Nicotiana tabacum L. were compared to the numbers of embryos and plantlets produced in cultured anthers excised at the optimal mitotic stage of development for anther culture. The Feulgen technique of staining embryos caused a considerable loss of grains from cultured anthers but this did not seriously affect the determination of the percentage of embryos present. In no instance did the numbers of embryos produced exceed the maximum number of S grains found, and the distributions of S grain and embryo frequencies in anthers were similar. In rare instances S grains which had undergone the first embryogenic division were observed in situ. The results indicate that all grains capable of embryogenesis are determined during early flower formation and that their number is not increased by in vitro culture.     

In vitro cytokinin binding to a particulate fraction of tobacco cells

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.Abbreviations BA N ⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - K_d equilibrium dissociation constant - R_t total concentration of binding sites In partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University     

Modulation of the number of membrane-bound auxin-binding sites during the growth of batch-cultured tobacco cells

We studied the modulation of the number of membrane-bound naphthaleneacetic acid (NAA)-binding sites during the growth cycle of tobacco cells in batch culture. Both cell number and specific NAA-binding increased exponentially, but at different rates and for different periods. This caused a characteristic modulation of the number of binding sites per cell during the growth cycle: During the first day of the lag phase this number decreased; in the exponential phase it rose markedly, and in the stationary phase it was constant.Abbreviations MES 4-morpholinoethanesulphonic acid - NAA 1-naphthaleneacetic acid     

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Hormonal control of tobacco protoplast nucleic acid metabolism during in vitro culture

Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosomal RNA, polyadenylated RNA, and proteins. These syntheses are essentially independent of the presence of hormones in the culture medium and are thus related to the ageing phenomenon induced by isolation from the plant and in-vitro culture. At all stages of culture and in all culture media, protoplasts incorporate low levels of thymidine into their DNA. However, the incorporation of considerable quantities of thymidine, indicative of the S phase, only takes place after 25–30 h and requires the presence of auxin and cytokinin.Abbreviations 6-BA 6-benzyladenine - 2,4-D 2,4 dichlorophenoxyacetic acid - DPC diethylpyrocarbonate - OD optical density; oligo-dT cellulose-oligothymidylic acid-cellulose - poly A⁺ RNA polyadenylated RNA - poly A^- RNA non-polyadenylated RNA - tRNA ribosomal RNA - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tris buffer Tris (hydroxymethyl)aminomethane - tRNA transfer RNA     

Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.)

A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4·10^-8 to 4·10^-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5·10^-8 to 5·10^-7 M), higher concentrations (5·10^-6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - i6Ade isopentenyladenine - i6Ado isopentenyladenosine     

Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia

The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 M Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 M Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 M Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.     

Selection of embryogenic pollen from cold-treated buds ofNicotiana tabacum var. badischer burley and their development into embryos in cultures

Summary By using density gradient centrifugation, employing 55% percoll and 4% sucrose as suspension medium, it is possible to select embryogenic pollen from buds after cold treatment at 10 °C for 8 or more days. These buds at the uninucleate stage of pollen were collected from plants grown in 8 hours photocycles at 18 °C and supplied with mineral salts. The embryogenic pollen are small, starch-free with a clear cytoplasm whereas large starch-filled ones are nonembryogenic. The embryogenic pollen regularly form embryos at a frequency of 2% on a mineral medium supplemented with glutamine, asparagine and sucrose at pH 6.5.These results demonstrate, for the first time, that it is possible to have embryos in appreciable frequencies in ab initio pollen cultures raised from cold treated anthers.On leave from University of Delhi.