Cell Cultures Of Chenopodium Rubrum Research Articles

Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation and exocytosis in heterotroph cell cultures of Chenopodium rubrum

We investigated the response of extracellular phosphatase to heat shock in heterotrophic Chenopodium rubrum L. cell cultures. Surprisingly, in contrast to the generally used acid phosphatase, an extracellular alkaline phosphatase showed the most sensitive response. This phosphatase was characterized as a marker for cellular stimulation by its high correlations with induced changes of extracellular pH: 10microM nigericin (correlation coefficient r=0.91), 100microM salicylic acid (r=0.84), heat shock 5min 37 degrees C (r=0.79), and heat shock after pre-treatment with 5microM fusicoccin (r=0.92) or 0.5% ethanol (r=0.90). Cellular stimulation was estimated with concentrations of acids and bases, yielding similar levels of pH change (0.5 pH) in cell-free supernatant: salicylic acid (200microM), benzoic acid (600microM), HCl (140microM), NaOH (100microM), and KOH (100microM). The Golgi apparatus inhibitor Brefeldin A (200microM) reduced the heat-shock-induced phosphatase (-33%). The pH optimum of heat-shock-induced phosphatase was 3; however, there the proportion of constitutive phosphatase was higher than at pH 8-9.5, indicating different pH dependence of constitutive and induced activity. Thus, heat-shock-induced phosphatase was characterized by alkaline activity with inhibitors (10microM molybdate: -52%, 2.5mM phosphate: -64%, 10microM ZnCl(2): -82%), substrates (2.5mM, tyrosine phosphate: 255pkat g(-1), p-nitrophenyl phosphate: 92pkat g(-1), serine phosphate: 0, threonine phosphate: 0), Hill coefficient (nH=1.4) indicating two binding sites, and the extent of heat-shock stimulation (p-nitrophenyl phosphate: +190%, tyrosine phosphate: +180%). SDS-PAGE showed a correlation of alkaline phosphatase with the heat-shock-induced release of highly N-glycosylated 53kDa protein, detected by peroxidase-labeled concanavalin A affinoblotting after endoglycosidase H treatment. The 53kDa protein showed no in-gel phosphatase activity after SDS-PAGE and regeneration treatment, in contrast to a putative dimer (105kDa).

PsbA Mutation (valine219 to isoleucine) inPoa annua resistant to metribuzin and diuron

The herbicide-binding region of the chloroplast psbA gene from a total of 20 biotypes of Poa annua L resistant and susceptible to metribuzin and diuron was selectively amplified using PCR. Sequence analysis of the fragment from six herbicide-resistant biotypes of P annua exhibited a substitution from valine to isoleucine at position 219 of the D1 protein encoded by the psbA gene. This is the same mutation as reported for Chlamydomonas and Synechococcus through site-directed mutagenesis and in cell cultures of Chenopodium rubrum L. To our knowledge this is the first report of a higher plant exhibiting resistance in the field to photosystem II inhibitors due to a psbA mutation other than at position 264. The existence of additional biotypes of P annua resistant to diuron or metribuzin but lacking mutation in the herbicide-binding region indicates that resistance to these herbicides can also be attained by other mechanisms. # 2000 Society of Chemical Industry

A new psbA mutation yielding an amino-acid exchange at the lumen-exposed site of the D1 protein.

In eight metribuzin-resistant photoautotrophic cell cultures of Chenopodium rubrum (Thiemann and Barz, 1994 a, b) sequence analyses of a part of the psbA gene coding for the photosystem-II D1 protein had revealed different double and triple mutations within the herbicide binding niche of the protein (Schwenger-Erger et al., 1993). Two pairs of the examined cell lines carried identical mutations within this part of the protein, although their growth performance and their herbicide resistance patterns were different, both at the cellular and the thylakoid level. Starting from the known part of the psbA gene we have amplified the remaining psbA sequences using inverse polymerase chain reaction. Thus the complete sequence of the coding part of the gene was elucidated. After sequence analyses we found an additional amino acid exchange at the position 184 (ile-->asn) of the D1 protein in cell line L1. Metabolic consequences of this mutation are discussed. Partial sequence analyses of the psbD gene of the herbicide resistant cell culture lines revealed no mutation within that part of the D2 protein, which is in direct contact with the D1 protein.

On the cytotoxity of oxidized phytosterols isolated from photoautotrophic cell cultures of Chenopodium rubrum tested on meal-worms Tenebrio molitor

Cell injury—as evoked by oxidative stress, insect attack or ageing—causes lipid peroxidation of polyunsaturated acids, e.g. linoleic acid. Hydroperoxides (LOOH) so produced transform fatty acids, terpenes and sterols to epoxides. We describe a bioassay to determine the cytotoxity of sterol oxidation products by measuring the mortality of meal-worms ( Tenebrio molitor) after injection of diluted test compounds. LOOH was tested in concentrations from 10 −5 M up to 10 −2 M and developed recognizable mortality (25%, 10 −2M, 23 hr). A direct relation between activity and concentration was observed. 5,6-Epoxides of phytosterols and derived 3,5,6-trihydroxysteranes have been isolated from Chenopodium rubrum cell cultures. Using 5,6-epoxycholesterol and 3,5,6-trihydroxycholestane as standards of known bioactivity, the toxicity of epoxides of sitosterol and stigmasterol, as well as the trihydroxy compounds thereof, was checked. The phytosterol derivatives are by a factor of five less active than the corresponding cholesterol oxidation products. 5,6-Epoxides and 5,6-chlorohydrins showed half of the activity of the corresponding 3,5,6-trihydroxyphytosterols. These developed highest cytotoxity (40% mortality in 5 × 10 −3 M solution after 23 hr). The activity of 5,6α-and 5,6β-epoxides was found to be equal. Metabolism and proposed biological function of oxidized phytosterols is discussed.

Oxidized phytosterols increase by ageing in photoautotrophic cell cultures of Chenopodium rubrum

Young and old cell cultures of Chenopodium rubrum were investigated for their content of phytosterol derivatives by gas chromatography-mass spectrometry. The ratio of the main phytosterol constituents, sitosterol, stigmasterol and campesterol (1.2:2.4:1.0) remained approximately constant, but their overall content (402.5 μg g −1 dry wt) decreased from 11 days to 8 weeks old cell cultures by about 64% (260.1 μg g −1 dry wt); in constrast the content of corresponding epoxides increased from traces at day 11 to 11.1 μg g −1 (dry wt) at the 8th week. A comparable increase was observed for 3,5,6-trihydroxysteranes (2.8 μg g −1 dry wt), day 11; 27.5 μg g −1 dry wt), 8 weeks). The content of sterols oxidized in position C-7 showed also an increase from 1.9 μg g −1 dry wt) (11 days) to 51.7 μg g −1 dry wt) (8 weeks). These results show that the increase of phytosterol oxidation products correlated with age of the cell culture. Therefore, the sterol epoxide content and related oxidation products may be used as a measure for ageing.

Photoautotrophic Chenopodium rubrum Cell Suspension Cultures Resistant against Photosynthesis-Inhibiting Herbicides II. Physiological and Biochemical Properties

Eight photoautotrophic cell cultures of Chenopodium rubrum, which are resistant against the photosystem II inhibitor metribuzin, were characterized for their growth parameters, chlorophyll content and photosynthetic capacity. Herbicide resistance of the eight lines results from different mutations in the D 1 protein of photosystem II, which is the target for different photosystem II inhibitors. In the presence of 10-5 ᴍ metribuzin the eight lines showed substantial growth reduction depending on the degree of resistance, and this effect is explained by a reduced electron transport in photosystem II. The impaired photosynthetic capacity of the green cells in the presence of high metribuzin concentrations, leads to compensation effects similar to shade accommodation of plants. Adaptation includes an increase of the chlorophyll content, a decrease of the chlorophyll a/b ratios as well as an increase of thylakoid stacking and cell number per unit fresh weight. In the absence of the herbicide photosynthetic electron transport is not impaired, as indicated by measurements of electron transfer rates in photosystem II and flash-induced reduction kinetics of P-700+. In summary the alterations of the D 1 protein of the eight cell lines do not result in a reduced electron transport in photosystem II.

Characterization of high-light-induced increases in xanthophyll cycle pigment and lutein contents in photoautotrophic cell cultures

The effect of prolonged high light treatment on xanthophyll pigment content was analysed in photoautotrophic cell cultures of Chenopodium rubrum and Marchantia polymorpha. Photoinhibitory effects of high light treatment were indicated by a reduction in F v/ F m (dark), which was accompanied by a partial de-epoxidation of the xanthophyll cycle pigment pool. Xanthophyll cycle pigments were found in grana thylakoids and stroma thylakoids and the epoxidation states were comparable in both thylakoid membrane subfractions. During high light treatment, the contents of the xanthophyll cycle pigments and lutein increased by de novo synthesis of these pigments. Increases were observed in grana thylakoids and stroma thylakoids. In the stroma thylakoid fraction, an enrichment of the 28 kDa apoprotein of light-harvesting complex IIb (LHCIIb) was found after high light treatment, but in unfractionated thylakoid membranes marked increases in the apoproteins of the dominant pigment-protein complexes could not be detected. In addition, the stoichiometry of pigment changes did not correspond to that of isolated pigment-protein complexes from untreated plants. Therefore we conclude that the ratio of xanthophyll cycle pigments to pigment-protein complexes was increased after prolonged light stress.

Intracellular Conversion of Malate and Localization of Enzymes Involved in the Metabolism of Malate in Photoautotrophic Cell Cultures of Chenopodium rubrum

Cells from photoautotrophic cultures of Chenopodium rubrum were fractionated for the isolation of purified chloroplasts and mitochondria. The subcellular localization of the enzymatic activities involved in the metabolism of malic acid was investigated. Highly purified chloroplasts were obtained from the protoplasts, whereas peroxisomes were still present in the mitochondrial fraction. NAD - and NADP-dependent malate dehydrogenase and malic enzyme activities were found in the mitochondrial and chloroplast fractions, respectively. Exogenously supplied [14C]labelled malate was metabolized by the photoautotrophic cell suspension, obviously to the greater part in mitochondria.

Hydroxycinnamic Acid Transferases in the Biosynthesis of Acylated Betacyanins: Purification and Characterization from Cell Cultures of Chenopodium rubrum and Occurrence in Some Other Members of the Caryophyllales*

An acyltransferase from cell cultures of Chenopodium rubrum was purified 515-fold with a 2.5 % recovery. This enzyme catalyzes the transfer of hydroxycinnamic acids from 1–0-hydroxycinnamoyl-/β-glucose to the C-2 hydroxy group of glucuronic acid of amaranthin (betanidin 5-O-glucuronosylglucose). The invivo products formed are celosianin I (4-coumaroylama-ranthin) and celosianin II (feruloylamaranthin). The enzyme can be classified as l-0-hydroxycinnamoyl-β-glucose: amaranthin O-hydroxycinnamoyl-transferase (EC 2.3.1.-). Its molecular weight was determined by gel filtration column chromatography to be ca. 69.5 kDa. Maximal rate of product formation was found to be at pH 5.6. The isoelectric point of the enzyme was at pH 4.7. The reaction temperature maximum was at 37 °C and the apparent energy of activation was calculated to be 44.5kJ mor−1. The enzyme showed a Vmax of 910pkat (mg protein)−1 with amaranthin as acceptor and feruloylglucose as acyl donor. The ratios of Vmax/Km values for sinapoyl-, feruloyl, caffeoyl- and 4-coumaroylglucoses were found to be 100:56:56:40. Donor competition experiments support the conclusion that one single enzyme is responsible for the ester formation with the different hydroxycinnamic acids. From the possible acceptors tested, only amaranthin (15S configuration) and isoamaranthin (15R) were esterified with Km values of 280 and 800 μM, respectively. Catalytic effectivity (Vmix/Km) was found at a relative ratio 15S:15R of 100:42. Betanin (betanidin 5-O-glucoside) and gomphrenin I (betanidin 6-O-glucoside) were not accepted. Some other acylated betacyanin-containing members of four families of the Caryophyllales were investigated and showed the same type of hydroxycinnamoyltransferase activity with 1-O-hydroxycinnamoylglucose as acyl donor, but with different acceptor molecule specificities.

Differential Regulation of Nuclear Genes during Blue Light‐dependent Chloroplast Differentiation in Cultured Plant Cells

AbstractNuclei capable of RNA synthesis were isolated from both dark‐grown and blue light‐irradiated cell cultures of Chenopodium rubrum. Analysis of the heterodisperse RNA sequences synthesized showed that the SSU and LHCP gene as well as early light‐induced genes were specifically transcribed. The relative transcription rate of their mRNAs increased with the onset of blue light irradiation reaching a maximum at about 12 h, then dropped to the dark level. A comparison with the steady‐state concentration of the same mRNAs in vivo revealed a substantial difference between the two groups of genes: For the three early light‐induced genes the in vitro transcription rate is in good accordance with the observed steady‐state level of their transcripts supporting the notion that the blue light‐dependent rapid and transient accumulation in vivo is caused by a temporary increase in the transcription rate of the corresponding genes. In contrast, for the SSU and LHCP gene, the changes in transcription rate do not follow those in steady‐state concentration; thus the observed steady increase of the latter in blue light cannot be explained as an enhancement in the transcription rates of both genes, but is a likely consequence of post‐transcriptional events.

Comparison of Light-Induced Stress Reactions in Susceptible and Paraquat-Tolerant Green Cell Cultures of Chenopodium rubrum L.

Summary A paraquat-tolerant line was isolated from green photomixotrophic cell cultures of Chenopodium rubrum L. Activity of enzymes of peroxide metabolism (catalase, glutathione reductase, peroxidase) was not significantly different in the tolerant cells. The sensitivity to symptoms of photodamage observed in suceptible control cells under high light intensity, such as photoinhibition, photoinactivation of catalase, or bleaching of chlorophyll, was not markedly mitigated in paraquat-tolerant cells. Paraquat-tolerance is not generally accompanied by cross resistance to oxidant stresses from other sources.

Blue Light‐Dependent Chloroplast Differentiation in Cultured Plant Cells: Evidence for Transcriptional Control of Plastid Genes*

AbstractA DNA‐protein complex capable of RNA synthesis (transcriptionally active chromosome, TAC) was isolated from plastids of dark‐grown and blue light irradiated cell cultures of Chenopodium rubrum. The transcriptional activity of TAC increased rapidly upon irradiation of dark‐grown cells with blue light. Analysis of the RNA synthesized showed that plastid rRNA genes as well as genes coding for membrane and stroma proteins were specifically transcribed; their quantities depended on the developmental stage of the plastids produced by blue light exposure. The in vitro transcription rate seems to reflect fairly well the in vivo situation insofar as its enhancement in blue light irradiated cells is in good accordance with the observed steady‐state level of plastid mRNAs. The results obtained support the notion that the blue light‐dependent accumulation of plastid mRNAs in vivo is caused by an increase in the transcription rate of the corresponding genes. Thus an alternative exists to the light‐induced transformation of etioplasts to chloroplasts in seedlings where primarily posttranscriptional and translational controls are effective.

Fructose-2,6-bisphosphate, metabolites and ?coarse? control of pyrophosphate: fructose-6-phosphate phosphotransferase during triose-phosphate cycling in heterotrophic cell-suspension cultures of Chenopodium rubrum

Experiments were carried out to determine whether pyrophosphate: fructose-6-phosphate phosphotransferase (PFP) catalyses the rapid recycling of triose phosphates that is found in the cytosol of heterotrophic cell cultures of Chenopodium rubrum L. (W.-D. Hatzfeld, M. Stitt, 1990, Planta, 180, 198-204). Oxygen uptake, carbohydrate turnover, fructose 2,6-bisphosphate (Fru2,6bisP), glycolytic intermediates, adenine and uridine nucleotides, pyrophosphate and the activity of PFP and glycolytic enzymes were monitored for 48 h after subculturing carbohydrate-depleted cells onto glucose. Immediately after transfer there was an increase in the amount of Fru2,6bisP, and of the hexose phosphate. The triose phosphates, fructose-1,6-bisphosphate and inorganic pyrophosphate increased gradually over the next 24 h. This was accompanied by a tripling in the extractable activity of PFP, but not of phosphofructokinase. The activity of fructose-1,6-bisphosphatase was 20-50fold lower than that of PFP. It is calculated that the activity of PFP is high enough to catalyse the observed rate of cycling between the triose phosphates and the hexose phosphates, based on the measured Vmax capacity of the enzyme, the known kinetic properties, and the measured levels of its reactants and Fru2,6bisP. The changes in the levels of Fru2,6bisP were not correlated with the rate of respiration. Instead, the rate of O2 uptake was inversely related to the phosphoenolpyruvate level, showing that pyruvate kinase or phosphoenolpyruvate carboxylase are regulating the use of glucose for respiration. There was also no relation between Fru2,6bisP, and partitioning to sucrose or starch. It is proposed that the main function of the cycle in these cells is to maintain high levels of inorganic pyrophosphate and triose phosphates, which are necessary for the remobilisation of sucrose and for biosynthesis in the plastid, and that 'coarse' and 'fine' control of PFP play an important role in regulating this cycle.

Turnover of O‐Glucosides of Dihydrozeatin and Dihydrozeatin‐9‐riboside During the Cell Growth Cycle of Photoautotrophic Cell Suspension Cultures of Chenopodium rubrum

SummaryThe cellular levels of O‐glucosides of 3H‐(diH)Z and 3H‐(diH)[9R]Z, the major short‐term metabolites of 3H‐(diH)Z having been exogenously supplied to photoautotrophically growing suspension cell cultures of Chenopodium rubrum, decreased significantly during further culture, irrespective of whether the cells were maintained in the stationary phase or were transferred to conditions restoring cell divison. Metabolism of both compounds was more pronounced during the active growth phase than during the stationary phase. The O‐glucosides were converted preferentially to polar compounds of as yet unknown nature, which were partly excreted into the medium. The cellular pools of both glycosides remained compartmented within the vacuole. In contrast to the O‐glycosides, the small cellular pools of the aglycones 3H‐(diH)Z and 3H‐(diH)[9R]Z maintained their level during the experimental period of 30 days. Small amounts of the glucosides, as well as of the aglycones, were recovered from the medium and could have resulted from the lysis of a few cells. The results demonstrate, for the first time, that O‐glucosides of cytokinins are not irreversibly deposited within the vacuole of plant cells but may serve to maintain a small, but more or less constant pool of extra‐vacuolar, presumably cytosolic, aglycones. (DiH)Z and its derivatives could be demonstrated to be endogenous cytokinins of Chenopodium rubrum suspension cultured cells occurring along with those of the isopentenyladenine and zeatin types.

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.

Lipids in plant tissue cultures. VII: Heterotrophic and mixotrophic cell cultures of Chenopodium rubrum

Mixotrophic cell cultures of Chenopodium rubrum were found to synthesize 5 to 33 times more monogalactosyldiacylglycerols and 5 to 16 times more digalactosyldiacylglycerols than heterotrophic ones. The monogalactosyldiacylglycerols and digalactosyldiacylglycerols from mixotrophic cultures contained higher levels of linolenic acid as compared to heterotrophic cultures. It is concluded that the active synthesis of these galactolipids with high levels of constituent linolenic acid is associated with the onset of photosynthesis in plant cell cultures, as is the case in intact plants.