No 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.

Methylotrophic bacteria inhabit the surface of plant organs, but the interaction between these microbes and their host cells is largely unknown. Protonemata (gametophytes) of the moss Funaria hygrometrica were cultivated in vitro under axenic conditions and the growth of the protonemal filaments recorded. In the presence of methylobacteria (different strains of Methylobacterium), average cell length and the number of cells per filament were both enhanced. We tested the hypothesis that auxin (indole-3-acetic acid, IAA), secreted by the epiphytic bacteria and taken up by the plant cells, may in part be responsible for this promotion of protonema development. The antiauxin parachlorophenoxyisobutyric acid (PCIB) was used as a tool to analyze the role of IAA and methylobacteria in the regulation of cell growth. In the presence of PCIB, cell elongation and protonema differentiation were both inhibited. This effect was compensated for by the addition of different Methylobacterium strains to the culture medium. Biosynthesis and secretion of IAA by methylobacteria maintained in liquid culture was documented via a colorimetric assay and thin layer chromatography. Our results support the hypothesis that the development of Funaria protonemata is promoted by beneficial phytohormone-producing methylobacteria, which can be classified as phytosymbionts.

The cytokinesis-related callose deposition in cell plates and juvenile cross walls of meristematic cells was investigated in the liverwort Riella helicophylla and seedlings of Arabidopsis thaliana. The b-1,3-glucan callose was detected by its specific staining properties with sirofluor and aniline blue by fluorescence microscopy. The photo-labile calcium antagonist nifedipine (NIF) exerted a specific promotive effect when the substance was exposed to light. The nitroso derivative of photolysed NIF was found to be the active compound which was responsible for the enhancement in callose deposition. The nitroso derivative was isolated after photolysis of NIF by UV light (365 nm) and its structure was verified with 1 H-nuclear magnetic resonance and infrared spectroscopy. The characteristic absorption maximum at 770 nm in dimethyl sulfoxide was employed to determine the concentration of the nitrosopyridine in solutions by use of the molar absorption coefficient of the isolated substance. In addition, the nitro derivative of nifedipine was prepared. This nitropyridine was ineffective with respect to the stimulation of callose deposition in dividing cells. The possible mechanism of this cytotoxic effect and its implications for symplastic growth in meristems is discussed.

The Ca(2+) indicator 7-chlorotetracycline has been shown to bind to a pore complex on both outer surfaces of all non-meristematic cells in the unistratose thallus of Riella ('chlorotetracycline-binding surface region'=CSR; Grotha, 1983, Planta 158, 473-481). Prolonged treatment of the thallus with 7-chlorotetracycline, 5-hydroxytetracycline, verapamil and desmethoxyverapamil induces the deposition of callose at the same region. The influence of various treatments on verapamil-induced CSR-callose was measured in situ by microfluorometry of aniline-blue-stained material. Callose deposition is maximal at 10(-4)M verapamil or 5·10(-5)M desmethoxyverapamil with 2·10(-4)M Ca(2+) or Mg(2+) in the medium. The reaction is completely inhibited at pH 5.5 and is optimal between pH 6.5 and 7.5. The production of CSR-callose is absolutely light-dependent with callose being first visible after 30 min of light. La(3+), ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid and amiprophosmethyl, antagonists of Ca(2+) functions, and 2-deoxy-D-glucose suppress the verapamil induction of CSR-callose. Furthermore the ionophores A 23187, valinomycin and monensin effectively block the reaction. The deposition of CSR-callose is diminished at increasing external osmolarity and is abolished at osmotic values that stimulate plasmolysis-callose. Wounding causes the formation of wound-callose but inhibits the induction of CSR-callose in cells of the wound edge. Nifedipine increases or prolongs callose synthesis in cell plates. The Ca(2+)-channel blocker diltiazem is completely ineffective. It is suggested as a working hypothesis that verapamil-induced CSR-callose synthesis is caused by a local change in membrane permeability, possibly as a consequence of the opening of Ca(2+) channels being involved in Golgi-vesicle mediated exocytosis (A. Kramer and H. Lehmann, 1986, Ber. Dtsch. Bot. Ges. 99, 111-121).

In juvenile walls of dividing cells of the liverwort Riella helicophylla the nitroso‐derivative of photolysed Nifedipine (a calcium antagonist) stimulates the deposition of callose. This enhanced biosynthesis of β‐1,3‐glucan can only be observed in the cell plate, the juvenile cell walls and the walls of adjacent cells. An immunocytological analysis of this effect revealed that no cortical microtubules occurred at the sites of callose deposition. The cells of the control displayed a normal distribution of cortical microtubules at the plasma membrane as long as no callose was deposited along the corresponding walls. In a second set of experiments, inhibitors of microtubule polymerization and depolymerization (amiprophosmethyl and taxol, respectively) were used. At low concentrations, these substances also caused a significant stimulation of callose deposition in the plane of cell division. Based on these findings, we propose a regulatory model of callose and cellulose biosynthesis that depends on the binding of the cellulose/callose synthase complex to cortical microtubules that may be mediated by unknown binding protein(s).

The Ca"-chelator CTC binds to a specific site on both outer surfaces of all non-meristematic cells of the unistratose thallus of Riella, known to be rich in anionic wall components and calcium, and induces there the deposition of callose. Structural changes in this region during prolonged CTC treatment have been followed by light and transmission electron microscopy. With fluorescence microscopy punctate structures can be detected after lOmin, which upon longer incubation in CTC develop into large vesicular bodies, surrounded by a circular structure. The aniline blue-derived fluorescence intensity of these structures is highest in cells of the extension growth zone.At the ultrastructural level a mosaic of numerous smooth-surfaced vesicles, presumably containing callose, initially appears subjacent to the plasma membrane. These vesicles swell and fuse with each other, forming ultimately a circular fusion profile with the plasma membrane. This complex of callose-forming vesicles is thought to develop from elements of the partially coated reticulum (PCR), based on the presence of coated vesiculation profiles on the callose vesicles and numerous aggregates of coated vesicles in their immediate vicinity. _~_ _ _ _ _ _ _ _ _~_ _ _~~After 30 min in CTC osmiophilic particles appear around these callose vesicles and at the cytoplasmic face of mitochondria. They are later (after 60 min) deposited in the periplasmic space between wall and plasma membrane and are also released into the surrounding medium. As judged by their reaction with FeC13, the osmiophilic particles appear to be phenolic in nature. We propose that upon binding of CTC a local increase of cytoplasmic calcium triggers callose synthesis in PCR-like compartments beneath the plasma membrane. However it remains to be shown as to why callose is synthesized exclusively in these intracellular compartments and not at the plasma membrane.

The unistratose thallus of the gemmaling of Riella helicophylla is divided into an apical growth lobe with the meristem, an intermediate pillar, and a basal rhizoidal lobe. This organization can be correlated with a distinct fluorescence pattern of wall and membrane after treatment with chlorotetracycline. Mature cells of the growth lobe are distinguished by two chlorotetracycline-binding surface regions (CSR) with a diameter of 6-12 μm in the middle of both outer cell surfaces. Meristematic cells are devoid of CSR. The same is true for cells of the pillar which elongate under low light intensity. Rhizoid initials have an enlarged CSR on the site where the rhizoidal tube will emerge, whereas the opposite cell surface lacks any chlorotetracycline fluorescence. With the beginning of the extension of the rhizoid, CSR remains as a ring around the base of the tube. Darkness, plasmolysis, and sulfhydryl reagents inhibit the fluorescence of CSR, whereas calcium antagonists in addition suppress the fluorescence of the rhizoids. Cytochemical methods demonstrate that sulfhydryl proteins and anionic polysaccharides are involved in adsorbing chlorotetracycline in this region. Surface electron-microscopic preparations reveal a local depression of the wall covered with amorphous material.