Epiphytic Bacteria Associated with the Bryophyte Funaria hygrometrica: Effects of Methylobacterium Strains on Protonema Development

Abstract: Bacteria represent a substantial fraction of the microorganisms that inhabit leaf surfaces. We collected samples of the moss Funaria hygrometrica (L.) in the field and analysed the epiphytes on the gametophyte by the agar impression method and scanning electron/fluorescence microscopy. On the phylloid surface numerous bacteria were detected, notably in the grooves between adjacent lamina cells. Methanol-ammonium salts agar surfaces impressed with isolated phylloids of green gametophytes resulted in the growth of methylotrophic colonies. Two Methylobacterium strains ( M. mesophilicum and M. sp., isolated from the Funaria phylloids) were found to simulate the well-known effect of cytokinin application on bud formation in Funaria protonemata. In addition, Methylobacterium inoculation promoted the growth of protonemal filaments. The significance of this novel Methylobacterium -land plant interaction is discussed.     

The control of chloroplast division in Funaria hygrometrica II. The effects of kinetin and indole-acetic acid on nucleic acids

Replication of chloroplast DNA is an initial stage in chloroplastdivision during regeneration of isolated moss leaves. Variousconcentrations and combinations of growth substances have beenused to ascertain their effects on initiation of cellular dedifferentiation.Results indicate that during early stages of dedifferentiationof cells of isolated leaves of the moss Funaria hygrometricaa .change in the concentration balance between indole-aceticacid (IAA) and kinetin is effective in controlling the initiationof DNA replication in nuclei and chloroplasts. One of the waysthe balance between growth substances changes in by rapid lossof IAA from the cut leaf surface after isolation of the leaf (Received January 8, 1971; )     

Calcium Ameliorates Effects of Lead in Protonema of Funaria hygrometrica Hedw.

Two-days-old in vitro grown protonemata of Funaria hygrometrica Hedw. were treated with a mixture PbCl₂ (4 M Pb²⁺) and CaCl₂ (16 M Ca²⁺) (Ca+Pb) for 48 h. The results were compared with the control: distilled water (H₂O) and the solution of PbCl₂ (4 M Pb²⁺) (Pb). Protonemata treated with Ca+Pb were longer and contained more cells than those treated with Pb. Moreover, a lower number of cells showed apical cell deformations typical for lead toxicity: swollen tips and wall thickenings at the apex. If deformations were present they were not as extended as in Pb. In comparison with the control, however, protonemata treated with Ca+Pb were shorter, contained a lower number of cells and some apical cells in this material were altered. It can be concluded that the presence of calcium partially neutralised toxic effects of lead in Funaria hygrometrica protonemata cells.     

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     

Aseptic cultivation of sapling trees for studies of nutrient responses with particular reference to phosphate

Plantlets of Betula verrucosa and B. pubescens, originally obtained from seeds surface sterilised in hydrogen peroxide, were grown on slopes of mineral nutrient agar, modified Ingestad, in plastic tubes kept at room temperatures (26 °C) with supplementary lighting. Cuttings taken from plants grown in this way rooted readily on media supplemented with 0–1 mg/1 naphthalene acetic acid and 10 g/1 sucrose. In replicated experiments to determine the optimal conditions for growth, the effects of varying salt concentrations, the addition of charcoal and changes in ventilation and amounts of phosphate were studied. It was found necessary to ‘confound’ variations attributable to the sources of seedlings/cuttings in the experimental designs. The method has facilitated the detection of clones in both B. verrucosa and B. pubescens, which, although growing equally well with large amounts of readily available phosphate, differed greatly when phosphate was in short supply. Low phosphorus ‘tolerant’ and ‘intolerant’ types were identified.     

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     

The quantitative response to cytokinin in the moss Funaria hygrometrica does not reflect differential sensitivity of initial target cells

Exposure to sufficient cytokinin induces the formation of buds from responsive cells in the protonema of Funaria hygrometrica. Initial perception of the phytohormone results in a Ca+2 cascade within minutes. A second cytokinin-mediated event occurs some days later, and converts incipient buds into stably committed buds. The concentration of exogenous cytokinin also regulates the total number of buds produced from a protonemal colony. This concentration-dependent production of buds has been thought to reflect differential sensitivity of target cells. Under that hypothesis, the regulation of bud number occurs during initial perception of hormone. This paper presents direct experimental evidence to the contrary and supports the alternate hypothesis that bud formation involves the gating of large numbers of responding cells by later events. Experiments transferring protonema between media with different levels of cytokinin show that the cytokinin concentration during the initial perception of cytokinin is unimportant in controlling bud number. Instead, bud number is found to be regulated by the concentration of exogenous cytokinin as incipient buds or bud initials become stably committed buds.     

Impact of prescribed burning on the nutrient balance of heathlands with particular reference to nitrogen and phosphorus

Question: Can prescribed winter burning compensate atmospheric nutrient loads for dry heathlands? What effects does prescribed burning have on nutrient balances, particularly as regards the limiting nutrients N and P? Location: Lueneburg Heath, NW Germany. Methods: In two burning experiments (in 10/15 year old Calluna‐stands) nutrient balances (for N, Ca, K, Mg, P) were calculated by analysing nutrient inputs (atmospheric deposition, ash deposition), nutrient stores (above‐ground biomass, organic horizon) and nutrient outputs (biomass combustion, leaching). Results: Atmospheric nutrient deposition amounted to 22.8 kg.ha^‐1.a^‐1 for N and < 0.5 kg.ha^‐1.a^‐1 for P. Nutrient stores in the above‐ground biomass were 95/197 kg.ha^‐1 for N and 5/13 kg.ha^‐1 for P (first/second experiment, respectively). From these stores 90/53% (for N) and 25/14% (for P) were removed by burning. Effects of leaching on nutrient balances were low. In the first two years after burning, leaching rates of N increased by about 4/6 kg.ha^‐1, whereas leaching rates of P did not change significantly. Input/output‐ratios showed that prescribed burning leads to positive nutrient balances for N, Ca and Mg in the long term. For example, the amounts of N removed by prescribed burning are equivalent to ca. five years of atmospheric inputs. Applied in ten‐year cycles, this measure alone cannot prevent N accumulation in the long term. Conclusion: Regarding 10/15 year old Calluna‐heaths, we assume that prescribed burning cannot compensate for atmospheric N inputs, thus making long‐term changes in the nutritional state inevitable. Therefore, prescribed burning should be applied in combination with high‐intensity management measures.     

Bud formation in Funaria hygrometrica: A comparison of the activities of three cytokinins with their ribosides

Summary The activities of the cytokinins benzyladenine (BA), 6-(3-methylbut-2-enylamino)purine (IPA) and zeatin in promoting bud formation in protonemata of Funaria hygrometrica (L.) Sibth. were compared to the biological activities of the corresponding ribosides. Cytokinins were found to have considerably higher hormonal activity as free bases than as ribosides. This observation will be discussed in relation to the chemical identity of the celldivision factor (CDF) isolated from crown gall tumors of Vinca rosea L.