Orientation of cell wall ß-glucan Synthases in plasma membrane vesicles

In cell-free experiments with membranes of tobacco callus UDP-Glc incorporation into β-glucans was influenced by UDP. With UDP in the assay, β-glucan formation was inhibited and almost completely vanished at concentrations higher than 10 mM. Similar levels of inhibition were achieved with ADP and GDP, whereas the respective nucleotide-triphosphates inhibited to a higher and the nucleotide-monophosphates to a lower degree. The inhibition with UDP was shown to be non-competitive. UDP increased the β-glucan formation by variable degrees, when it was enclosed within membrane vesicles. The stimulation of β-glucan biosynthesis with enclosed UDP occurred mainly in inside out (ISO) plasma membrane vesicles. UDP in the assay inhibited the β-glucan formation as well in right side out (RSO) membrane vesicles as in ISO vesicles. Inhibitors of phospholipid glycosylation like amphomycin, tunicamycin and bacitracin inhibited β-glucan formation in the presence of 0.1% digitonin. Bacitracin without digitonin the in vitro β-glucan biosynthesis. The kinetics for inhibition by amphomycin and bacitracin were compared with kinetic parameters of different concentrations of digitonin. The results were discussed with respect to the two possibilities of enzyme/substrate interactions

Cell wall regeneration by Nicotiana tabacum protoplast: Chemical and biochemical aspects

Protoplasts isolated enzymatically from the mesophyll of tobacco leaves (Nicotiana tabacum L.) were cultured in a defined liquid medium. The composition of the cell wall polysaccharides (CWP) and of the extracellular polysaccharides (ECP) synthesized after 1–4 days of culture was investigated by gas-liquid chromatography (GLC) in comparison with that derived from tobacco mesophyll and tobacco tissue cultured on solid agar or in suspension. The cell wall of regenerating protoplasts was composed mainly of non-cellulosic polysaccharides with glucose predominating (65%) and a content of only 5% cellulose whereas the cell walls of leaves and cultured tissue contained 60% and 45% cellulose, respectively. The cell walls of leaves, callus and suspension cultured cells showed differences in their arabinose/ xylose ratio. Galactose, arabinose and uronic acids were the main constituents in the ECP of regenerating protoplast and of suspension cultured cells indicating the secretion of pectic material into the culture medium. The labelling pattern of polysaccharides synthesized by protoplasts after culture with [U-14C]glucose or [U-14C]sucrose was in accordance with the chemical composition. No label of the generally used osmoticum mannitol was incorporated into polysaccharides. Radioactivity derived from myo-[3H]inositol was transformed into uronic acids and pentoses, but not incorporated into the different hexoses. Simultaneous feeding of [U-14C] glucose and unlabelled myo-inositol reduced incorporation of radioactivity into uronic acids and pentoses. Hence, the myo-inositol pathway is operative in cell wall regenerating tobacco protoplasts

The influence of phytohormones on growth, organ differentiation and fructan production in callus of Symphytum officinale L.

Callus derived from Symphytum officinale L. regenerants was cultured in the presence of various phytohormones. The growth rate of callus was stimulated by all phytohormones at various concentrations. With 1-naphthaleneacetic acid no organ differentiation could be observed. With indole-3-butyric acid at low concentrations only roots were formed, whereas 6-benzylaminopurine, kinetin and zeatin at various concentrations induced either root or shoot formation or the simultaneous regeneration of both. Minor amounts of fructans were formed at high 6-benzylaminopurine-, zeatin- and at all indole-3-acetic acid-concentrations. The concentration of 1-naphthaleneacetic acid had no influence on the fructan content. Highest rates of fructan synthesis occurred at low zeatin-concentrations up to 1.5 mg/l. Only zeatin at all concentrations induced the synthesis of polyfructans, whereas appreciable amounts of oligofructans were formed under the influence of all other phytohormones

Fructan Synthesis in Tissue Cultures of Symphytum officinale

Tissue cultures originating from different organs i.e. leaves, leaf-stalks, ovaries, anthers, and roots of Symphytum officinale were initiated under various growth conditions and subcultured several times to give the first callus generation. From all these calli, whole plants could be regenerated which again were used for the preparation of tissue cultures resulting in the formation of the second callus generation. The different calli and the regenerated plants were analyzed with respect to the fructan-synthesizing capacity. Only calli derived from the leaves of the original plant synthesized fructan whereas calli derived from ovaries, anthers, and roots, which are known to contain large amounts of fructan, were not capable of synthesizing fructan. The regenerated plants obtained from the first callus generation showed ability for fructan synthesis only if the originating callus synthesized fructan. The calli of the second generation, which were prepared from fructan-containing leaves and roots of regenerated plants, showed the capacity for fructan formation. The calli of the second generation obtained from leaves and roots of regenerated, fructan-free plants were not able to synthesize this specific reserve polysaccharide. From these data it can be concluded that the calli of the first generation prepared from roots, ovaries, and anthers have lost their ability for fructan synthesis. Calli initiated from leaves and leaf-stalks preserved the capacity for fructan formation even after many calli generations and regeneration to entire plants. Different phytohormones used in the tissue cultures had only a slight effect upon the fructan formation. An influence of light on fructan synthesis could not be detected