Cytokinin effect on branching and bud formation in Funaria

Cytokinins have two different effects on protonemata of Funaria hygrometrica. They induce branching of unbranched caulonemata and bud formation. Branching occurs after treatment with pico-molar concentrations of cytokinins whereas bud formation requires micro-molar concentrations. Both processes are therefore independently stimulated by cytokinins.Abbreviation BA N⁶-benzyladenine     

Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca²⁺-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca²⁺ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca²⁺ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca²⁺ indicate a localized rise in intracellular free Ca²⁺ concentration brought about by cytokinin action.Abbreviations BA 6-benzyladenine - CTC chlorotetracycline - ER endoplasmic reticulum - NPN N-phenyl-1-naphthylamine     

Bud formation inFunaria: Organelle redistribution following cytokinin treatment

Summary Cytokinin stimulates caulonemata ofFunaria to undergo an asymmetric division leading to the gametophore. The earliest detectable event is a small protuberance at the distal portion of the cell accompanied by the reorganization of the underlying organelles into a polarized distribution reminiscent of a tip growing cell. Dictyosomes and associated vesicles accumulate in the protuberance directly beneath the plasma membrane with mitochondria subjacent to the vesicular layer. Endoplasmic reticulum lies beneath the mitochondrial zone directly above the large central vacuole, while chloroplasts are outside the bud. As development continues the bud elongates causing the outer cell wall to exfoliate. During the above events the nucleus migrates toward the bud site concomitant with an increase in the number of microtubules between the nucleus and the base of the outgrowth. Nucleoli, extruded from the nucleus during a previous division, persist as diffuse fragments within the protuberance. Upon reaching the bud site, division occurs with the developing phragmoplast being initiated distal to the caulonema tip cell. The former polarized distribution of the cytoplasm is altered as mitochondria, chloroplasts and small vacuoles become evenly dispersed throughout the cytoplasm; dicytosomes and endoplasmic reticulum occupy a cortical position. These events indicate a change from 2-D tip growth to 3-D diffuse growth. To quantify the ultrastructural changes associated with bud formation we performed a morphometric analysis of cells in various stages of budding. The relative volumes of dictyosomes and vesicles adjacent to the bud apex decrease during bud development coincident with an increase in these organelles in lower portions of the cytoplasm. Mitochondria and chloroplasts follow this same pattern although their highest relative volumes initially are 4 m from the bud apex and outside the bud site, respectively. These data, as well as density profile topographic maps for vesicle fractions, support the contention that cytokinin induces a change in morphological symmetry and polarity in the fine structure ofFunaria.     

Induction of budding on chloronemata and caulonemata of the moss,Physcomitrella patens,using isopentenyladenine

The bud-inducing effect of the cytokinin N⁶-(²-isopentenyl)-adenine (i⁶-Ade) was examined in the moss Physcomitrella patens growing in liquid culture. Under these conditions, buds could be induced on chloronemata as well as on caulonemata. By application of i⁶-Ade, bud-formation was accelerated in both types of tissue. The number of buds, their size and their site of development were dependent on the concentration of the cytokinin in the range of 10^-7 M to 10^-5 M. Moreover, the percentage of caulonema cells increased with a cytokinin concentration of 10^-5 M. These results indicate that chloronema cells may also function as target cells for exogenous cytokinins. The composition of proteins from caulonemata and chloronemata of two different species (P. patens and Funaria hygrometrica), grown on solid medium were compared. No differences could be detected between the protein patterns of caulonemata and chloronemata of the same species while between the two species the differences were obvious.Abbreviations i⁶-Ade N⁶-(²-isopentenyladenine) - Da dalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Forced calcium entry and polarized growth of Funaria spores

We have used both steady electric fields, and gradients of the divalent ionophore, A23187, to control the point at which rhizoids emerge from spores of the common moss Funaria hygrometrica. The spores were grown in a medium containing calcium nitrate as the only major salt. Spores tend to form rhizoids towards the positive electrode, with a half maximal response to a difference of 4–8 mV across each cell. They also tend to form rhizoids towards the end of higher ionophore concentration in response to A23187 gradients. Both of these responses are the same at pH 5.5 and 8.0. Our tentative explanation is that Funaria spores tend to form rhizoids where most calcium enters. However, the point of chloronema emergence is scarcely affected by steady fields of up to 45 mV/cell. Moreover, when steady fields are applied across already developed rhizoids or chloronemata, their subsequent growth is directed towards the negative electrode in both cases, with rhizoids giving a 50% response at only 3—5 mV/cell, and chloronemata being less responsive.From Tsung-Hsien Chen's Ph.D. thesis, Purdue University     

Observations on dividing plastids in the protonema of the moss Funaria hygrometrica Sibth.

The process of division was investigated in the different types of plastids found in the tip cell of the protonema of Funaria hygrometrica Sibth. There were no structural changes in the envelope membranes of any of the plastid types during the initial stage of division. As the process of constriction advanced, thylakoids were locally disintegrated and sometimes starch grains in the isthmus were locally dissolved. In the isthmus, tightly constricted plastids were characterized by an undulating envelope and an increasing number of vesicles. After three-dimensional reconstruction of electronmicrographs a distinct filamentous structure was observed in the plane of division outside the plastid but close to the envelope. At different stages of division the constricted regions were partly surrounded by one or a few filaments. The roundish plastids in the apical zone were accompanied by single microtubule bundles, and the spindle-shaped plastids in the cell base were surrounded by single microtubules and microtubule bundles. A model of co-operation between microtubules and the filamentous structure in the division process is discussed.A preliminary report was presented at the Tagung der Deutschen Botanischen Gesellschaft und der Vereinigung für Angewandte Botanik, Hamburg, September 1986     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.

Three-week-old protonemata of Funaria hygrometrica Hedw. cultivated in Petri dishes tolerate slow drying (24 h to complete dryness) but not rapid drying (1h to complete dryness). Slowly dried mosses show, on a dry-weight basis, a sixfold increase in abscisic-acid (ABA) contents during the drying process. Rehydrated, slowly dried protonemata have the ability to tolerate subsequent rapid drying. When ABA is added to three-week-old protonemata at a concentration of 10 M for 16 h, tolerance to rapid drying is induced. These data indicate that the induction of drought tolerance in Funaria hygrometrica is mediated by ABA. Mosses treated with ABA loose their water as fast as controls do; therefore, ABA does not act via reduced water loss. However, induction of synthesis of new proteins by ABA may form an important part of the drought tolerance because 10 M cycloheximide inhibits the ABA-mediated tolerance to rapid drying.Abbreviations ABA abscisic acid - CHI cycloheximide - DW dry weight - FW fresh weight - RWL relative water loss This work was supported by grants from the Deutsche Forschungs-gemeinschaft and by a NATO fellowship awarded to R.M. Ros Espin.     

In vitro cytokinin binding to a particulate cell fraction from protonemata of Funaria hygrometrica

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [³H]benzyladenine (BA) to a 13,000–80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10^-8 and 10^-4M. [³H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [³H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [³H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000–80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption.Abbreviations BA N⁶-benzyladenine - IPA 6-(3-methyl-2-enylamino)purine - 9-MeBA N⁶-benzyl-9-methyladenine     

Characterisation of a temperature-sensitive gametophore over-producing mutant of the moss, Physcomitrella patens

Summary A mutant of the moss, Physcomitrella patens, was isolated which was temperature-sensitive for the production of gametophores. At 17° C this mutant, designated ove 409, produced normal leafy shoots. At 24° C ove 409 produced many abnormal buds characteristic of bud-over-producing (ove) mutants. ove 409 produced an intermediate phenotype at 21° C. The cytokinin levels in the culture medium of this mutant, the wild-type and a cytokinin overproducing mutant, oveA78, were measured by combined gas chromatography mass spectrometry at the permissive and nonpermissive temperatures. Production of cytokinin was found to be affected by temperature in all strains; the change in phenotype of ove 409 correlated with the production of N⁶-(²-isopentenyl) adenine. Complementation analysis was performed using this mutant by protoplast fusion. ove 409 was found to be in the same complementation group as a previously isolated ove mutant, oveA78.     

Tip-localised H+-fluxes and the applicability of the acid-growth hypothesis to tip-growing cells: Control of chloronemal extension in Funaria hygrometrica by auxin and light

The role of tip-localised H⁺ secretion in regulating chloronemal tip growth in the moss Funaria hygrometrica Hedw. was investigated. pH was monitored with pH microelectrodes placed close to the cell surface while the rate of extension growth was manipulated by illumination and by the application of indole-3-acetic acid. Growth stimulations were accompanied by acidification of the external solution; this acidification was most pronounced at the growing tip. The timing and extent of acification external to the tip correlated well with the magnitude and time course of growth stimulations. The maintenance of both growth and H⁺ efflux under CO₂-free conditions indicated that neither photosynthetic nor respiratory CO₂ metabolism were involved. Artificially acidifying the nutrient solution rapidly but transiently stimulated elongation in both white light and darkness. Furthermore, the stimulation of elongation caused by white light was inhibited if the nutrient solution was buffered strongly near neutrality. We conclude that the acid growth hypothesis is applicable to tip growth in Funaria and that light and exogenous indole-3-acetic acid act at least in part by stimulating localised H⁺-ion efflux.Abbreviations D darkness - IAA indole-3-acetic acid - WL white light     

Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D₂O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.Abbreviations DMSO dimethylsulfoxide - CB cytochalasin B Dedicated to Prof. Dr. A. Pirson on the occasion of his 70. birthday     

Correlation of electrical current influx with nuclear position and division inFunaria caulonema tip cells

Summary Ionic currents around caulonema tip cells of the filamentous protonema of the mossFunaria hygrometrica were examined using a nonintrusive vibrating microelectrode to map electrical current before and during mitosis. Tip cells in interphase generate inward electrical currents that are maximal at the nuclear region. These currents remain concentrated over the nucleus as it migrates forward maintaining a constant distance from the growing tip. Just prior to mitosis this inward current increases twofold. During mitosis and cytokinesis current at the nuclear zone increases to four times the resting level and fluctuates, falling to zero after cell plate fusion with parental walls. The locus of outward current could not be dectected. These results suggest that plasma membrane ion currents may regulate both nuclear positioning and subsequent temporal and spatial control of cell division.     

Androgenesis in Citrus aurantifolia (Christm.) swingle

By means of gas chromatography-selected ion monitoring-mass spectrometry using an isotope-dilution assay with 4,5,6,7-tetradeutero-indole-3-acetic acid as the internal standard, indole-3-acetic acid has been estimated to be present in aseptically cultured gametophytes of wild-type Physcomitrella patens (Hedw.) B.S.G. at a level of 0.075 g g^–1 dry weight or 2.1 ng g^–1 fresh weight.Abbreviations IAA indole-3-acetic acid - d₄IAA 4,5,6,7-tetra-deutero-indole-3-acetic acid - [¹⁴C]IAA indole-3-[2-¹⁴C]-acetic acid - GC-SIM-MS gas chromatography-selected ion monitoring-mass spectrometry     

Partial purification and initial characterization of phytochrome from the mossAtrichum undulatum P. Beauv. grown in the light

The extraction and partial purification of phytochrome from light-grownAtrichum undulatum P. Beauv., a chlorophyllous moss, is described. Polyethyleneimine and salt fractionation followed by hydroxyapatite and Affi-gel-blue chromatography were used to separate phytochrome from chlorophyll, and to purify the pigment. All steps were performed in the presence of Triton X-100 which improved the yield by a factor of about three. The protein has a molecular weight some-what larger than that ofAvena phytochrome (124 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. It cross-reacts with a monoclonal antibody against phytochrome from etiolated corn (Zea) and a polyclonal antibody against phytochrome from etiolated oat (Avena), and its photoreversibility is similar to that of phytochrome from greenAvena.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavinmononucleotide - PMSF phenylmethylsulfonylfluoride - P_r(P_fr) red(far-red)-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Phoneutria nigriventer venom: a cocktail of toxins that affect ion channels

1. We review the pharmacological actions of toxins present in the venom of the aggressive spider Phoneutria nigriventer.2. This venom is rich in toxins that affect ion channels and neurotransmitter release. Voltage-gated sodium, calcium, and potassium channels have been described as the main targets of these toxins.3. In addition to these classical actions Phoneutria toxins have also been shown to affect glutamate transporter.4. It is expected that molecular genetics in addition to biochemical, biophysical and pharmacological approaches will help to further define Phoneutria toxins and their mechanisms of action in the near future.     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Normal development and fine structure

Summary The regular branching of theFunaria caulonema filaments is partly related to rhythms in nuclear and cell division. The formation and development of the branches were studied by light and electron microscopy with particular attention directed to the distribution of microtubules and the polar organization of the cytoplasm. The new side branch breaks through the wall of the mother cell. The site of branch development is determined by the position of the nucleus of the mother cell. In protonemata which grow in vertically placed Petri dishes gravity influences the position of nuclei and side branches, and also the direction of oblique cross walls in the caulonema filaments to a certain extent.     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside