Culture Growth Cycle Research Articles

Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of tobacco Bright Yellow 2

The ascorbate (ASC) and glutathione (GSH) metabolisms were studied in cultured Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells. TBY-2 cells were found to be endowed with L-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), an enzyme that converts L-galactono-γ-lactone into ASC. Cellular fractionation of TBY-2 protoplasts indicated that this enzyme is exclusively localised in mitochondria and associated to the membrane fractions. During the growth cycle of TBY-2 cell culture, GLDH transiently increased, reaching the maximum value on the third day of culture, at the beginning of the exponential phase, when the cell proliferative activity was also higher. Similar behaviour has been observed for ASC and GSH contents. The activities of ascorbate peroxidase (APX) (EC 1.11.1.11), ascorbate-free radical reductase (AFRR) (EC 1.6.5.4), dehydroascorbic acid reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) also transiently raised. However, the scale of the increases varied being about 4-fold for APX and AFRR, 2-fold for DHAR and more than 11-fold for GR. The behaviour of the ASC and GSH recycling enzymes allowed TBY-2 cells to maintain both dehydroascorbic acid and glutathione disulphide at low levels, even under conditions of high ASC and GSH utilisation. The relationship between the ASC and GSH metabolisms during the growth cycle of TBY-2 cell suspension cultures is also discussed.

Temporal Correlation of Tyramine Metabolism with Alkaloid and Amide Biosynthesis in Elicited Opium Poppy Cell Cultures : In Honour of Professor G. H. Neil Towers 75th Birthday

Aromatic amines are involved in the biosynthesis of many plant secondary metabolites, including benzylisoquinoline alkaloids and hydroxycinnamic acid amides. Incorporation of tyramine and dopamine into both the antibiotic alkaloid sanguinarine and hydroxycinnamic acid amides was shown to occur in elicitor-treated opium poppy cell cultures. A temporal correlation was demonstrated between the biosynthesis of alkaloids and amides, the accumulation of tyrosine\\dopa decarboxylase (TYDC) and berberine bridge enzyme (BBE) mRNAs, and the induction of tyramine hydroxycinnamoyl transferase (THT) activity. The induction of TYDC mRNAs occurred most rapidly, whereas the subsequent activation of BBE mRNAs and THT activity correlated with the synthesis and accumulation of sanguinarine and amides, respectively. In pulse-labeling experiments, a maximum of 1.2 and 0.6% of exogenous 14C-tyramine and 14C-dopamine, respectively, was incorporated into sanguinarine after elicitor treatment of cell cultures. In contrast, a maximum of 24 and 35% of exogenous 14C-tyramine and 14C-dopamine, respectively, was incorporated into an acid-insoluble fraction of the cell wall after elicitor treatment. Insolubilized radioactivity was released from cell walls after hydrolytic extraction and recovered mostly as hydroxycinnamic acid amides and free amines. The elicitor-induced incorporation of 14C-amines into both sanguinarine and cell walls decreased during the cell culture growth cycle. The prolonged induction of THT activity in elicited cultures, coupled with high and constitutive peroxidase activity, suggests that the observed decrease in cell wall labeling 5 h after the addition of elicitor was due to hydroxycinnamoyl-CoA or H2O2 limitation.

Changes in functional properties of mitochondria during growth cycle of Arabidopsis thaliana cell suspension cultures

Composition and properties of Percoll-purified mitochondria isolated from Arabidopsis thaliana cell suspension cultures were studied at various stages of the growth cycle. During the lag phase (2 days) mitochondria exhibited high lipid/ protein ratio, high double bound index and very low oxidative rate. After day 2 and up to day 4 for NADH (exponential phase), or to day 7 (beginning of the senescence) for succinate as substrates, the oxidation rate in state 3 increased while oxidation rate in state 4 remained low. Consequently, mitochondria appeared more and more coupled over the whole time of the growth cycle. Along with the constant oxidation rate in state 4, characteristics of membranes remained similar in terms of polypeptide pattern, proton conductance or fluorescence anisotropy although unsaturation of membranous fatty acids increased significantly during the growth cycle. All these results indicated that oxidation rate in state 3 increased continuously while basic properties of mitochondrial membranes remained similar during the growth.

Cell cultures of Rauwolfia sellowii: growth and alkaloid production

Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 0.2 mg l-1 kinetin and 30 g l-1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h-1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC.

Transient Accumulation of Glycine Betaine and Dynamics of Endogenous Osmolytes in Salt-Stressed Cultures of Sinorhizobium meliloti

The fate of exogenously supplied glycine betaine and the dynamics of endogenous osmolytes were investigated throughout the growth cycle of salt-stressed cultures of strains of Sinorhizobium meliloti which differ in their ability to use glycine betaine as a growth substrate, but not as an osmoprotectant. We present (sup13)C nuclear magnetic resonance spectral and radiotracer evidence which demonstrates that glycine betaine is only transiently accumulated as a cytoplasmic osmolyte in young cultures of wild-type strains 102F34 and RCR2011. Specifically, these strains accumulate glycine betaine as a preferred osmolyte which virtually prevents the accumulation of endogenous osmolytes during the lag and early exponential phases of growth. Then, betaine levels in stressed cells decrease abruptly during the second half of the exponential phase. At this stage, the levels of glutamate and the dipeptide N-acetylglutaminylglutamine amide increase sharply so that the two endogenous solutes supplant glycine betaine in the ageing culture, in which it becomes a minor osmolyte because it is progressively catabolized. Ultimately, glycine betaine disappears when stressed cells reach the stationary phase. At this stage, wild-type strains of S. meliloti also accumulate the disaccharide trehalose as a third major endogenous osmolyte. By contrast, glycine betaine is always the dominant osmolyte and strongly suppresses the buildup of endogenous osmolytes at all stages of the growth cycle of a mutant strain, S. meliloti GMI766, which does not catabolize this exogenous osmoprotectant under any growth conditions.

Two δ 1 -pyrroline-5-carboxylate dehydrogenase isoforms are expressed in cultured Nicotiana plumbaginifolia cells and are differentially modulated during the culture growth cycle

δ1-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography. The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl− at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K m values for the two substrates, P5C and NAD+, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected, while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth. Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed.

Cell-cycle kinetics of Friend erythroleukemia cells in a magnetically shielded room and in a low-frequency/low-intensity magnetic field.

This work was undertaken to compare the behavior of Friend erythroleukemia cells in a solenoid, where the magnetic field was 70 microT at 50 Hz (plus 45 microT DC of Earth) with that of the same cells in a magnetically shielded room, where the magnetic field was attenuated to 20 nT DC and 2.5 pT AC. The control laboratory magnetic field corresponded to 45 microT DC and a stray 50 Hz field below 0.2 microT. The culture growth cycle of cells maintained inside the solenoid was slightly accelerated compared with that of cells maintained outside the solenoid (P < .05). This stimulation probably depended on sensitivity of cell cycle to a magnetic field, because, inside the solenoid, the percentage of G1 cells slightly increased during the culture growth cycle, whereas that of S cells slightly decreased. Acceleration of growth was detected soon after exposure of the cultures to the solenoid field, and growth did not change further if the action of this field continued for a long time, accounting for adaptation. The solenoid field also caused a small increase of cell survival without influencing cell volume. By contrast, the culture growth cycle of cells maintained inside the magnetically shielded room was slightly decelerated compared with that of cells maintained outside the room (P < .05). The essential absence of any field inside the magnetically shielded room also caused a small increase of cell volume, whereas, during the culture growth cycle, the percentage of G1 cells decreased, and that of S cells increased. The majority of these events did not change in cells induced to differentiate hemoglobin through dimethylsulfoxide.

Properties of the 5- enol-pyruvyl-shikimate-3-phosphate synthase isoforms isolated from maize cultured cells

Two isoforms of the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase, previously purified from maize cultured cells and both found to be functionally located in the plastid while showing a different pattern of expression during the culture growth cycle, were characterized with respect to physical and functional properties. A high degree of similarity was found as to their structural features, with the only exception of a slight difference in molecular mass. Both enzyme activities were extremely susceptible to the inhibition brought about by the herbicide glyphosate, and not subjected to feed-back regulation by aromatic amino acids or shikimate pathway intermediates. A more pronounced difference was evident in thermal stability, catalytic efficiency as judged from the comparison of catalytic constants, affinities for the two substrates and activation energy values. The isozyme detectable in actively proliferating cells, when the plant cell demand for aromatic amino acids increases, proved to be the more stable and efficient. Data are consistent with the hypothesis of an isoform-based mechanism of enzyme level modulation in plant aromatic metabolism.

Regulation of DNA repair and replication in proliferating FL cells

In this paper, we show that the rate of total DNA synthesis increases twice during the Friend erythroleukemia (FL) cell culture growth cycle. The first increase takes place at the threshold of the logarithmic (early) phase of growth, while the second peak takes place in the mid-logarithmic (late) phase. This bi-phasic pattern can be reproduced in both normal and dimethylsulfoxide (DMSO)-differentiating cells, using either [3H]-thymidine or [14Cl-methyl-L-methionine as precursors for DNA labelling. In normal cells, the non-replicating strands incorporate some radioactivity originating in [3H]-thymidine, essentially in the early phase, while their labelling is insignificant in the late phase. In UV-irradiated cells, the mechanism yielding repair patches (RPs) contained by the same non-replicating strands is highly facilitated in the earlier stage in comparison with its functioning at the later stage. In contrast, the [3H]-thymidine labelling of the semiconservatively newly replicating strands is insignificant at the early stage and more substantial later, as expected. In UV-irradiated cells, the slightly decreased [3H]-thymidine labelling of replicating strands is probably explained by the concomitant small percentage of cell death. The chronology of the optimal rates of early repair and later replication is quite well confirmed by a bi-phasic regulation of the total DNA poly-merase (pol) activity: while a peak of N-ethylmaleimide (MalNEt)-resistant pols activity (accounting for base excision-repair) is seen early on, a peak of MalNEt-sensitive polα activity (accounting for replication) is found later. DMSO strikingly inhibits both the rate of the early base excision-repair (either exploiting [3H]-thymidine or [14C]-methyl-L-methionine) and the early MalNEt-resistant pols activity. It does not, however, inhibit the late replication and the late MalNEt-sensitive polα activity, when hemoglobin (Hb) synthesis is heavily induced. The time-shift of late replication in DMSO-differentiating cells is probably explained by a delay of theS-phase, as also suggested by the net decrease in the amount of DNA per nucleus during the second half of the culture growth cycle.

Effects of nitrogen concentration and cold temperature on DSP-toxin concentrations in the dinoflagellate Prorocentrum lima (Prorocentrales, Dinophyceae).

The diarrhetic shellfish poisoning toxin-producing dinoflagellate, Prorocentrum lima, isolated from Nova Scotian waters, contained both okadaic acid (OA) and dinophysistoxin-1 (DTX-1) throughout its growth cycle in culture; maximum concentrations of toxins and highest OA/DTX-1 ratios occurred during the stationary phase. Cells of P. lima survived 0 degrees C for 5 weeks and recovered when brought to a higher temperature. During the cold period, some cell damage probably occurred with concomitant losses of toxins to the medium. Nitrogen concentration in the medium was used to limit growth or stress the cells physiologically, and when growth was limited, increases in toxin associated with the cells were recorded. The relative amounts of okadaic acid were always greater than dinophysistoxin-1, but the significance of these ratios remains to be determined.

Growth and toxin production of the toxic dinoflagellate Pyrodinium bahamense var. compressum in laboratory cultures.

Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties).

The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion- and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. The last step resulted in two well-separated activities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial activity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. Two EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified plastid preparations demonstrated a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed.

Constitutive expression of extracellular peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes during the growth cycle of grapevine suspension cell cultures

The constitutive expression of peroxidase isoenzymes which are capable of oxidizing 4-hydroxystilbenes was studied during the growth cycle of suspension cell cultures from grapevine (Vitis vinifera L. cv. Monastrell) berries. the results showed that the growth of suspension cell cultures is accompanied by the constitutive expression of the peroxidase isoenzymes HSPrx 1 and HSPrx 2, previously characterised by their properties for oxidizing 4-hydroxystilbene, the expression of these isoenzymes being characteristic of Monastrell vines brought to a resistant state. However, although the differential catalytic properties of these grapevine isoenzymes are also shown by the horseradish peroxidase, no immunological relationships have been found between the peroxidases from either source.

Induction of a wheat Em promoter by ABA and optically pure ABA analogs in white spruce (Picea glauca) protoplasts

In white spruce (Picea glauca) protoplasts, abscisic acid (ABA) and optically pure ABA analogs induced expression of a reporter gene under regulation of a wheat ABA‐responsive promoter. A fusion of a 650 bp promoter fragment from the wheat Em gene promoter and the Escherichia coli uidA sequence encoding β‐glucuronidase (GUS) was linked in the plasmid pBM 113Kp. Expression of the Em‐uidA fusion varied among 6 white spruce genotypes. Protoplasts from 4‐day‐old embryogenic suspension cultures gave the highest GUS activity relative 10 other stages in the 7‐day growth cycle of suspension cultures. Racemic ABA [R.S‐(±)‐ABA] induced a significant increase of protoplast GUS activity over background at a concentration of 1 × 10−5M, but maximum GUS activity was found at 1 × 10−3M, ABA stereochemistry had a significant effect on gene expression. The natural isomer of ABA [S‐(+)‐ABA] was an effective inducer at a concentration as low as 1 × 10−7M, but a concentration of greater than 1 × 10−4M was required for induction by [R‐(—)‐ABA]. Moreover, analogs with the same configuration at C‐l1 as that of natural ABA were more effective for induction of expression from the Em‐uidA. insert at 1 × 10−4M than were their enamiomers. Plasnud pBI511. carrying the chloramphenicol acety] transferase (CAT) gene driven by the constitutively expressed, tandemly duplicated cauliflower mosaic virus 35S promoter, was co‐electroporated with pBM113Kp for monitoring Ihe influence of addition of exogenous ABA or ABA analogs on heterologous gene expression in protoplasts. CAT activity was not significantly affected by the presence or absence of ABA or the analogs used.

Methyltransferase activity in Allanthm altissima cell suspension cultures

Enzymes for the methylation of 1hydroxycanthin-6-one and a series of coumarins have been isolated from Ailanthus altissima cell suspension cultures. The coumarin methyltransferases methylate aesculetin to scopoletin and isoscopoletin, but not scopoletin, to scoparone. Fraxetin was methylated to isofraxidine but not to fraxidine and only fraxidine was methylated to 6,7,8-trimethoxycoumarin. These enzymes were studied throughout the culture growth cycle with two cell lines: 1, which produced 1-methoxycanthin-6-one as the major alkaloid and 2, in which canthin-6-one was the major alkaloid.

Time-course of accumulation of free extra- and intracellular amino acids in Achillea millefolium cell suspension cultures

A time-course study of free intra- and extracellular amino acids accumulation in cell suspension cultures of Achillea millefolium was performed by GC and GC-MS. The total amount of intracellular free amino acids diminished early in the lag phase and the first days of the exponential phase. Early in the stationary phase there was a great increase in the amount of intracellular free amino acids, which decreased afterwards in the late stationary phase. The amount of extracellular amino acids was rather small when compared to the amount of free intracellular amino acids and attained its maximum in the stationary phase. From the 17 amino acids identified during the growth cycle of the cell suspension cultures of A. millefolium, glutamic acid and glutamine were the two main free intracellular amino acids, while glutamic acid was the dominant amino acid detected extracellularly.

Determination of cell-wall porosity by microscopy: walls of cultured cells and pollen tubes

In previous studies the porosity of the plant cell wall has been estimated to lie between 3.5 and 6 nm, depending on the cell type investigated and the method used. Morinda citrifolia cells from cultures at various stages of growth were incubated for 60 min with a colloidal gold sol (CGS) of size range 1.9-7.9 nm. Penetration of the cell wall by these gold particles was limited to the log phase of the culture growth cycle, with maximum uptake occurring in early log phase. The size of particles observed within the early log phase cells ranged from 2.5 to 5.7 nm. Since the cell wall was not, as in other methods, disrupted or altered, we conclude that the wall pores are up to 5.7 nm mean diameter at this early stage, but decrease rapidly as the culture ages

Alkaloid metabolism in Tabernaemontana divaricata cell suspension cultures

O-Acetylvallesamine is the main alkaloid found in both medium and cells of the Tabernaemontana divaricata cell suspension culture investigated. [ 15N] O-Acetylvallesamine was added to the culture medium. Rapid exchange between biomass and culture medium was shown by the rapid distribution of labelled compound over the two compartments. The occurrence of biosynthesis throughout most of the investigated period was confirmed by the observed dilution of the labelled compound. Transformation, calculated from the decrease in the total amount of I 15N] O-acetylvallesamine, was also observed and occurred mainly during the stationary phase of the batch cell culture growth cycle.

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.)

The expression of calmodulin mRNA and protein were measured during a growth cycle of carrot (Daucus carota L.) cells grown in suspension culture. A full-length carrot calmodulin cDNA clone isolated from a lambdagt10 library was used to measure steady-state calmodulin mRNA levels. During the exponential phase of culture growth when mitotic activity and oxidative respiration rates were maximal, calmodulin mRNA levels were 4- to 5-fold higher than they were during the later stages of culture growth, when respiration rates were lower and growth was primarily by cell expansion. Net calmodulin polypeptide synthesis, as measured by pulse-labeling in vivo with [(35)S]methionine, paralleled the changes in calmodulin steady-state mRNA level during culture growth. As a consequence, net calmodulin polypeptide synthesis declined 5- to 10-fold during the later stages of culture growth. The qualitative spectrum of polypeptides synthesized and accumulated by the carrot cells during the course of a culture cycle, however, remained largely unchanged. Calmodulin polypeptide levels, in contrast to its net synthesis, remained relatively constant during the exponential phases of the culture growth cycle and increased during the later stages of culture growth. Our data are consistent with increased calmodulin polypeptide turnover associated with periods of rapid cell proliferation and high levels of respiration.