The lipids in photoautotrophic and heterotrophic cell suspension cultures of Chenopodium rubrum

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.     

Growth characteristics of hormone and vitamin independent photoautotrophic cell suspension cultures fromChenopodium rubrum

Summary This communication reports the photoautotrophic growth of hormone and vitamin independent cell suspension cultures ofChenopodium rubrum. The transfer of cells from stationary growth into fresh culture medium results in a high protein formation, followed by an exponential phase of cell division, whereas the onset of rapid chlorophyll formation is delayed for 4 days. At the stage of most rapid cell division there is no net synthesis of starch and sugar. When the cells enter stationary growth, there is a progressive accumulation of chlorophyll, sugar, and starch.Photoautotrophic cell cultures assimilate about 80–90 mol CO₂/mg chlorophyll X hour. Dark CO₂ fixation is about 3.7% to 2.2% of the light values during exponential and stationary growth, respectively. As shown by short-term¹⁴CO₂ fixation, CO₂ is predominantly assimilated through ribulosebisphosphate carboxylase via the Calvin pathway. There is a significant increase in the¹⁴C label of C₄ carboxylic acids in exponentially dividing cells as compared to cells from stationary growth. Thein vitro activity of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase is almost equal during exponential cell division. A decrease in cell division activity is accompanied by a significant change in the specific activities of both carboxylation enzymes. In non dividing cells from stationary growth the activity of ribulosebisphosphate carboxylase is greately enhanced and that of phosphoenolpyruvate carboxylase is reduced, documenting the development of carboxylation capacities typical for C₃-plants.The experimental results provide evidence that phosphoenolpyruvate carboxylase activity might be regulated by ammonia and could be involved in anaplerotic CO₂ fixation which supplies carbon skeletons of the citric acid cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - FDP fructose bisphosphate - F-6-P fructose-6-phosphate - G-6-P glucose-6-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenolpyruvate - RuDP ribulosebisphosphate     

Expression of a sugar-transporter gene family in a photoautotrophic suspension culture of Chenopodium rubrum L.

Photoautotrophic suspension-culture cells of Chenopodium rubrum L. were shifted to mixotrophic growth by adding glucose to investigate whether the activities of plant sugar transporters, as well as the expression of the corresponding genes, are regulated in response to sugars. The rate of d-glucose uptake was shown not to be affected by mixotrophic growth in the presence of d-glucose. The polymerase chain reaction (PCR) technique was applied to amplify cDNA and genomic fragments from monosaccharide-carrier genes. Seven members of a monosaccharide-carrier family were identified of which three were found to be expressed in the suspension-culture cells. The expression of the monosaccharide-carrier genes was independent of the presence of d-glucose.Abbreviation PCR polymerase chain reaction We would like to thank Michaela Bittner, Rainer Ehneß and Monika Kammerer for skillful technical assistance and S. Buchhauser and H. Hallmer for photographic work. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 43) and by Fonds der Chemischen Industrie.     

Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida Benth.

Photoheterotrophic and photoautotrophic cell suspension cultures were raised from a callus tissue derived from a Morinda lucida Benth. plant (Rubiaceae). The cultures were characterized with regard to fresh weight, dry weight, cell number, pH, chlorophyll and quinoid natural products. The amount of lipoquinones (phylloquinone, -tocopherol, plastoquinone, ubiquinone) isolated from the photoautotrophic cultures matched the amount detected in an intact leaf. Anthraquinone glycosides which are found in the roots of Morinda plants were not present in the photoautotrophic culture. The photoheterotrophic culture contained only trace amounts of these pigments. Abundant anthraquinone synthesis was observed when photoautotrophic and photoheterotrophic suspension cultures were transferred into darkness, provided sucrose was present in the medium. Induction of synthesis of anthraquinone pigments coincided with a rapid disappearance of lipoquinones from the culture. Thus, in the suspension culture, photoautotrophy correlates with lipoquinone synthesis and heterotrophy correlates with anthraquinone synthesis. This reflects the situation in the intact plants where lipoquinones are chloroplast-associated whereas anthraquinones occur in the roots.Abbreviation HPLC high-performance liquid chromatography     

Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, , determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5–2·10^–6 cm s^–1 bar^–1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.     

Dynamics of limiting cell wall porosity in plant suspension cultures

Changes in the limiting porosity of cell walls, i.e. the size limit for permeation of neutral molecules through the wall, were studied in several higher-plant cell-suspension cultures. For this purpose, samples of biomass fixed at different cultivation times were investigated using a method based on size-exclusion chromatography of polydisperse dextrans before and after equilibration with the extracted cell clusters. In suspension cultures of Chenopodium album L., Dioscorea deltoidea Wall. and Medicago sativa L., the mean size limit (MSL; critical Stokes' radius for exclusion of neutral polymers from half of the intracellular space) was found to vary between 2.4 and 3.8 nm. It decreased significantly during transition from the growth phase to the stationary phase. In the case of the C. album culture this change was found to be irrespective of whether sucrose in the medium was completely depleted at the end of the growth phase or not. The MSL was kept constant for long periods of the stationary phase if cell viability was maintained by repeated sucrose supplement. In a suspension strain of Triticum aestivum L., the MSL of cell wall permeation was comparatively small (1.75 nm) and remained constant during all cultivation phases. Relations between limiting porosity and cell wall growth, loss of pectic compounds to the medium, cross-linking activities and cell wall stiffening are discussed. Received: 19 December 1996 / Accepted: 23 April 1997     

Pectins as mediators of wall porosity in soybean cells

The non-invasive technique of fluorescence redistribution after photobleaching was employed on soybean (Glycine max (L.) Merr.) root cells grown in suspension culture to examine macromolecular transport across plant cell walls. Using both fluorescently derivatized dextrans and proteins of graded size, a functional range of diameters for putative trans-wall channels was determined to be 6.6–8.6 nm. A mild treatment with pectinase apparently enlarged the channels, without adversely affecting cell viability, enabling significantly larger molecules to pass through the wall. Treatment of the cells with cellulysin or protease did not have this enlargement effect. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Fl-dextran fluorescein-derivatized dextran - FRAP fluorescence redistribution after photobleaching - kDa kilodalton     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Pharmacology of the SV channel in the vacuolar membrane of chenopodium rubrum suspension cells

Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of –100 mV, the SV channel showed half-maximal inhibition with 20mm tetraethylammonium (TEA), 7 m 9amino-acridine, 6 m (+)-tubocurarine, 300nm quinacrine, and 35 m quinine, respectively. The SV channel is also blocked by charybdotoxin (20nm at –80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol. 61:687–708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.We are grateful to Prof. F. Dreyer and Dr. J. Beise from the Pharmacology Department of the Justus-Liebig-Universität Giessen for continuous interest and helpful suggestions. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Be 466/21-5) and the Bundesminister für Forschung und Technologie, Bonn.     

Photosynthetic characteristics of photomixotrophic and photoautotrophic cell suspension cultures ofChenopodium rubrum

Summary Cell suspension cultures ofChenopodium rubrum have been grown for more than 2 years photoautotrophically with CO₂ as sole carbon source. Average increase in fresh weight is appr. 600% within 14 days. The chlorophyll content of photoautotrophic cells (200 g/g fresh weight) is much higher than of photomixotrophic cells (50 g/g fresh weight). The photosynthetic activity of the cells (190 moles CO₂×mg^–1 chlorophyllXh^–1) is comparable to the values found with intact leaves. As shown by short-term¹⁴CO₂ photosynthesis, both, the photomixotrophic and the photoautotrophic cell suspension cultures assimilate CO₂ predominantly via the Calvin pathway.Major differences were found with cells from either exponential or stationary phase of growth with regard to differential labelling of 3-phosphoglyceric acid, malate, sucrose and glucose/fructose.In vitro measurements of carboxylation reactions only partially corroborate our findings with¹⁴CO₂ incorporation. The ratio of ribulosebisphosphate to phosphoenolpyruvate carboxylase activity is 4.7 for leaves of C.rubrum, 1.2 for photoautotrophic cells during stationary growth and 0.5 for cells during exponential growth phase, however, 0.18 was found for photomixotrophic cells. Though the¹⁴CO₂ incorporation into 3-phosphoglyceric acid is clearly higher than into malate, thein vitro activity of phosphoenolpyruvatecarboxylase is 2–6 fold higher than that of ribulosebisphosphate carboxylase. We postulate that anaplerotic reactions of the tricarboxylic acid cycle are involved in the regulation of phosphoenolpyruvate carboxylase.Abbreviations 2,4-D didilorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - fr. w. fresh weight - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PPO 2,5-diphenyloxazole - PEP phosphoenolpyruvate - RuBP nbulosebisphosphate     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum

[³H]Dihydrozeatin supplied to photoautotrophically growing cell suspension cultures of Chenopodium rubrum was rapidly taken up and metabolized by the cells. The predominant metabolites in extracts of the cells were [³H]dihydrozeatin-O-glucoside and [³H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [³H]dihydrozeatin and [³H]dihydrozeatin riboside, which were both present to a minor extent in cell extracts, were both present to a minor extent in cell extracts, were localized predominantly outside the vacuole. Analysis of the culture medium at the end of the 36-h incubation period showed that there had been an efflux of [³H]dihydrozeatin metabolites out of the cells. Whereas [³H]dihydrozeatin riboside was found to be the major extracellular [³H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [³H]dihydrozeatin could be detected in the medium. The differential compartmentation of [³H]dihydrozeatin metabolites found with the C. rubrum suspension-culture system is discussed with respect to possible mechanisms governing the metabolism of cytokinins in plants cells.Abbreviations (diH)Z dihydrozeatin - (diH) [9R]Z 9--D-ribofuranosyl dihydrozeatin - HPLC high-performance liquid chromatography - ODS octododecyl silica - PEP phosphoenolyruvate     

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20–70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20–140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure.Abbreviations FDA fluorescein diacetate - FITC-20 kDa, FITC-40 kDa, FITC-70 kDa dextrans fluorescein-derivatized 20-kDa, 40-kDa, and 70-kDa dextrans - IgG immunoglobulin G - kDa kilodalton Paramjit K. Gharyal wishes to thank the Nitrogen Availability Program at Michigan State University for financial support. We also thank Edwin de Feijter of Meridian Instruments for technical assistance in performing the confocal measurements. This work was supported by a grant from the U.S. — Israel Binational Agricultural Research and Development Fund (BARD project No. US-1384-87).     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of -l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)₄ — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H₂O₂ was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.Abbreviations CM-cellulose carboxymethyl-cellulose - FPLC fast protein liquid chromatography - HF hydrogen fluoride - HRGP hydroxyproline-rich glycoprotein - Hyp hydroxyproline - V_c retention volume - TCA trichloroacetic acid - TFA tri-fluoroacetic acid This work was supported by a A.F.R.C. postdoctoral assistantship to Michael D. Brownleader. We thank Dr. Anthony K. Allen (Department of Biochemistry, Charing Cross and Westminster Hospital, London, UK) for performing the amino-acid analysis and Mrs. Margaret Pickering (Department of Biochemistry, Royal Holloway) for performing the peptide-sequence analysis of extensin. We also express our gratitide to Dr. A. Mort (Oklahoma State University) for performing the HF-deglycosylation of extensin.     

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-¹⁴C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.     

Floral and growth responses in Chenopodium rubrum L. to an extract from flowering Nicotiana tabacum L.

Flowering of Chenopodium rubrum seedling plants was obtained in continuous light after application of fractions of a partially purified extract from leaves of flowering Maryland Mammoth tobacco (Nicotiana tabacum). The stage of flowal differentiation was dependent on the age of the Chenopodium plants used for the bioassay. Apices of plants treated with the extract at the age of four or seven days showed an advanced branching of the meristem or the beginning of formation of a terminal flower; treatment with the extract of plants 12 d old resulted in rapid formation of flower buds in all assay plants. Non-treated control plants kept in continuous light remained fully vegetative. The effects of the extract on flowering were associated with pronounced growth effects. Floral differentiation was preceeded by elongation of the shoot apex. Extension of all axial organs occurred, while growth of leaves, including leaf primordia, was inhibited. The pattern of growth after application of the flower-inducing substance(s) did not resemble the effects of the known phytohormones, but showed some similarities to growth changes resulting from photoperiodic induction of flowering.