Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

Establishment and characterization of American elm cell suspension cultures

Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A₇₀₀ and media conductivity. Combined cell growth data for all experiments within a genotype showed relatively low variances and between-genotype contrasts during repeated passages showed no significant differences. Subculturing exponentially growing cells at 8–14 day intervals, within readily measured parameters of media conductivity (4.95–4.2 mmhos) and cell concentration (≥ 1.4 A₇₀₀), consistently resulted in repeatable profiles of elm cell growth and minimized lag phase. Culture cells were essentially homogeneous after 5 subculture passages and their overall appearance was stable. We conclude that the described procedure resulted in consistent cultures suitable for elicitor treatment experiments. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of accumulated phosphate on culture growth and formation of cinnamoyl putrescines in medium-induced cell suspension cultures of Nicotiana tabacum

The inorganic phosphate of the liquid nutrient medium was completely taken up by freshly inoculated cells of Nicotiana tabacum L. within the first 2 d of culture. Thus intracellular ortho-phosphate concentrations of approx. 0.06 M were accumulated, which upon growth of the cultures were diluted by cell division and subsequent cell growth. Cells from different stages of the growth cycle containing progressively decreasing levels of phosphate were transferred to a phosphate-free medium which normally stimulates the formation of cinnamoyl putrescines. The resulting accumulation of these compounds was inversely correlated with the intracellular phosphate level, whereas a direct linear relationship in the phosphate concentration was found with further growth in the phosphate-free medium.Abbreviations 2,4D 2,4-dichlorophenoxyacetic acid - FW fresh weight - DW dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.     

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.Contribution Number 217.     

Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L.

Thalli of the haploid liverwort Marchantia polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1–4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.     

Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis

A rapidly growing and fine-textured cell line, NB2P, was established from Japonica rice cultivar Nipponbare and characterized in this study. Addition of casein enzymatic hydrolysate (2 g/L) and pectinase (0.005%) to the suspension medium resulted in a 2-fold-increased rate of cell growth and reduced aggregation. Remarkably, the medium and conditions described here resulted in growth leading to a 9-fold increase in fresh weight 7 d after subculture. High-quality, well-dispersed nuclei were obtained from this NB2P cell culture. Fluorescence-activated cell sorting (FACS) analysis of the isolated nuclei showed a clear separation of each cell cycle phase in both small- and large-scale preparations. On the basis of representative data from the nuclei fraction in the G1 phase, purity of the sorted and recovered nuclei was higher than 98%. The studies described here demonstrate that NB2P culture can be a powerful tool for studying many important plant processes, including DNA replication and cell cycle-related pathways.     

A cell suspension ofLinum flavum (L.) in phosphate limited continuous culture

A cell suspension ofLinum flavum was grown in phosphate limited continuous culture at two different growth rates. Energy metabolism (respiration), coniferin and lignin production and overall biomass composition were analysed, in order to establish the relations between growth, maintenance and secondary metabolism. The ATP turnover rate was higher in the faster growing cultures, corresponding with a higher energy requirement. The coniferin production was not directly correlated with the growth rate, indicating the possibility of high production at high growth rates. Steady states grown under identical conditions showed different characteristics that may have evolved during pre-culture time.     

Changes in peroxidases in the suspension culture of Rubus fruticosus during growth

The growth parameters of a cell suspension culture of Rubus fruticosus L. were determined over a culture period including exponential growth, stationary phase and a glucose starvation period at the end of the normal culture cycle. Peroxidase activities were measured in the cytoplasm, in the cell wall, and in the culture medium by the guaiacol assay. There is a relationship between the activity found in the spent medium and the dry matter mass of the cells during the exponential growth. In the three compartments a bimodal repartition of peroxidase activities was observed, with the two peaks at day 4 and day 26, respectively. This suggests that the first peak corresponds to actively dividing cells whereas the second is associated with senescence, or stress due to starvation. Fractionation of the peroxidases from the culture mediuim revealed the presence of two sets of cationic isoenzymes, with minor amount of anionic peroxidases. Interestingly, the second peak of cationic enzymes which was of weak intensity at day 10 of the culture, becameprevalent at day 26. This indicates that not only the total amount of peroxidases varies as a function of culture time, but also that the nature of the peroxidases secreted into the medium changes during growth.Abbreviations DW dry weight - FW fresh weight - MV medium volume - SV suspension volume - BSA bovine serum albumin     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Binding of colchicine to a soluble fraction of carrot cells grown in suspension culture

The binding of [³H]colchicine to soluble component prepared from carrot (Daucus carota L.) cells in suspension culture was assayed by the diethylaminoethyl(DEAE)-cellulose powder method. The binding activity was very labile and the time course of the binding indicated that the colchicine-bound complex was also unstable. The reaction was enhanced by vinblastine but lumicolchicine had no effect. The optimum temperature for the reaction was 30° C, and the colchicine binding constant was calculated to be 3.5·10⁴ l mol^–1 at 30° C.     

Dissimilation curves as a simple method for the characterization of growth of plant cell suspension cultures

A simple non-invasive method for the characterization of growth of a plant cell suspension in a single culture flask is given. The dissimilation of sugars by a cell-culture causes a loss of weight of the contents of the culture flask, and can therefore be used to follow the growth in that single culture flask. Because a correction for water evaporation is necessary, accurate results can only be obtained when a stable closure is used (e.g. Silicosen T-type plugs). The dissimilation curves obtained in this way were correlated to the concentration of sugars in the medium, the dry weight and the fresh weight. From these correlations the amount of intracellularly stored carbohydrates could be estimated. Rate constants for CO₂-diffusion were determined for different types of closure. These values allowed the estimation of CO₂ levels inside the culture flasks from the dissimilation curves (CO₂ release curves). The dissimilation curves obtained using this method can easily be related to other types of growth curves. Different growth-phases can be clearly distinguished, e.g. lag-phase, exponential growth-phase and stationary-phase.     

The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota

Anthocyanin-containing vacuoles were isolated from protoplasts of a cell suspension culture of Daucus carota. The vacuoles were stable for at least 2 h as demonstrated by the fact that they showed no efflux of anthocyanin. The uptake of radioactively labelled anthocyanin was time-dependent with a pH optimum at 7.5, and could be inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. Furthermore, the transport was specific, since vacuoles from other plant species showed no uptake of labelled anthocyanin, and strongly depended on acylation with sinapic acid, as deacylated glycosides were not taken up by isolated vacuoles. Hence, it is suggested that the acylation of anthocyanin, which is also required for the stabilization of colour in vacuoles, is important for transport, and that acylated anthocyanin is transported by a selective carrier and might be trapped by a pH-dependent conformational change of the molecule inside the acid vacuolar sap.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - PVP polyvinylpyrrolidone - TLC thin-layer chromatography     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17–20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.