Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

The elicitation of phytoalexins by Ca2+ and cyclic AMP in carrot cells

Addition of calcium ionophore A23187 or dibutyryl cyclic AMP (dBcAMP) to carrot (Daucus carota L.) cell culture induced the production of 6-methoxymellein, a phytoalexin of carrot, in a dose-dependent manner. Several reagents known to suppress the cytoplasmic calcium concentration appreciably inhibited elicitor-promoted phytoalexin production in carrot cells. The addition of elicitor to the carrot culture caused a rapid increase in the intracellular level of cyclic AMP. Treatments of the cells with theophylline or cholera toxin stimulated the biosynthesis of 6-methoxymellein even in the absence of elicitor. These observations suggested that Ca²⁺ and cyclic AMP participate as second messengers in the regulation of 6-methoxymellein production in cultured carrot cells. Addition of verapamil to carrot cell culture markedly inhibited 6-methoxymellein production when it was added within 30 min after elicitor-treatment of the cells, but no inhibitory effect was observed after 60 min. The results suggest that these messengers function in an early stage of the elicitation process. Carrot cells which were previously treated with verapamil accumulated only small amounts of 6-methoxymellein following the addition of dBcAMP. In contrast, cells incubated initially with dBcAMP accumulated the phytoalexin at levels comparable to the control when verapamil was added to the culture.     

Carrot phytoalexin inhibits Ca2+, calmodulin-dependent protein phosphorylation in carrot cells

Phosphorylation of soluble proteins obtained from cultured carrot cells was monitored by measuring the incorporation of ³²P from [³²P]ATP into the trichloroacetic acid insoluble fraction. The reaction was stimulated by Ca²⁺ and calmodulin, and inhibited by a carrot phytoalexin, 6-methoxymellein. 6-Methoxymellein also inhibited the Ca²⁺, calmodulin-dependent phosphorylative activation of NAD:quinate oxidoreductase (EC 1.1.1.24) partially purified from the carrot cells. The inhibitory effect of 6-methoxymellein was reduced when the reaction mixture contained a high concentration of calmodulin.     

Lipids of Daucus carota cell suspension cultures

In carrot cell suspension cultures greater amounts of phospholipid were detected than in carrot root material, but the major phospholipid classes were the same in both materials, and their fatty acid composition was very similar. In contrast to the cultured cells, no significant amounts of free fatty acids and monoglycerides, as well as diglycerides, could be detected in the carrot root. The fatty acid composition of the major lipid classes from cell cultures is reported for the first time in this report. The degree of unsaturation was higher in triglycerides and phospholipids than in free fatty acids. In a study of phospholipid biosynthesis, [³H]-glycerol was shown to be incorporated into 4-phospholipids (PE, PC, PG, PS⁷) to different extents. The highest specific activity was observed in PC and PG. Five molecular species were isolated from each of the 4 phospholipids and analyzed by GC-MS and LSC.     

Regulated expression of plant tRNA genes by the prokaryotic tet and lac repressors

The prokaryotic tet operator (tetO) sequence was inserted at positions upstream and downstream of sequences encoding the Arabidopsis thaliana tRNA _AUC ^Lys or tRNA _AUC ^Trp suppressor tRNAs, and tRNA expression in carrot protoplasts was measured by translational suppression of a nonsense codon in a luciferase reporter gene. Regulation of tRNA expression by the tetracycline repressor (tetR) occurred from genes with the tetO inserted at position –1 (for the tRNA _AUC ^Trp gene), or at positions –2, –6 and –10 (for the tRNA _AUC ^Lys gene), and repression reached 90%. The inducer tetracycline (Tc) restored tRNA expression. Similarly, carrot protoplasts transfected with human tRNA _AUC ^Ser genes containing the lac operator (lacO) in their 5-flanking sequence with or without the lac repressor (lacI) gene, conditionally expressed tRNAs which suppressed the luciferase reporter. Up to 30-fold repression occured by the lactose repressor when lacO was located at position –1 of the tRNA _AUC ^Ser coding sequence. In the presence of the inducer isopropyl--thiogalactoside (IPTG), repression was relieved. These results demonstrate that sequences flanking tRNA genes can strongly influence tRNA expression in plants, and in a conditional fashion when bound by inducible proteins.     

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [³H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [³⁵S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.     

An ultrastructural study of the fusion of cultured amphibian cells with higher plant protoplasts

Summary CulturedXenopus cells have been induced to fuse with carrot suspension cell protoplasts using PEG at high pH in the presence of high Ca²⁺. Ultrastructural observations confirm unambiguously that the fusion bodies seen by light microscopy are animal/plant cell heterokaryons. The csytoplasmic events occurring in theseXenopus/carrot fusion products during the first 48 hours of culture provide evidence for their viability. Some of the factors influencing the formation and subsequent survivalin vitro of interkingdom heterokaryons are discussed.     

Tissue specific alcohol dehydrogenase isozyme variation in carrot: Whole plant versus in vitro cultured cells

Summary Tissue specific qualitative variation in the zymmogram profile of alcohol dehydrogenase in carrot is described. Extracts made from roots and petiole tissues of the plant exhibit three and two bands of activity, respectively. No alcohol dehydrogenase (ADH) activity is detectable in lamina extracts. Similar extracts from carrot cells cultured in vitro exhibit five bands of ADH activity. However, when the same cell line is cultured in medium either lacking auxin (2,4-D) or Zn⁺⁺, no ADH activity can be observed on the gels.     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.     

Breakdown of Phosphatidylinositol during the Elicitation of Phytoalexin Production in Cultured Carrot Cells

Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. An inhibitor of Ca²⁺-phospholipid dependent protein kinase (protein kinase C), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor. Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca²⁺-mediated processes including calmodulin.     

Effects of chloramphenicol on plant cells: Potential as a selectable marker for transformation studies

The growth of tobacco and carrot cells in suspension cultures and on solidified medium was strongly inhibited by the antibiotic chloramphenicol. This effect was irreversible. The drug had no effect on [³H]-uridine incorporation but inhibited protein synthesis and induced striking morphological alterations in intoxicated cells. O₂ consumption was also reduced in treated cells. Tobacco and carrot cells were unable to inactivate chloramphenicol by enzymatic acetylation.     

Use of an electrochemical technique to study plasmamembrane redox reactions in cultured cells of Daucus carota L.

By exploiting the electron transfer reactions of ferricyanide-ferrocyanide at a gold electrode, an electrochemical technique was devised and successfully employed for the measurement of extracellular ferricyanide reduction or ferrocyanide oxidation by carrot (Daucus carota L.) cells grown in suspension culture. This technique eliminated problems of cell damage and insensitivity encountered when these activities are measured spectrophotometrically in magnetically stirred cell suspensions. Cells harvested from the mid-exponential phase of culture growth catalysed a rapid reduction of ferricyanide which was accompanied by H⁺ extrusion and was stimulated by ethanol. These cells also oxidised ferrocyanide, which was not associated with H⁺ extrusion. These observations can be explained on the basis of a plasmamembrane located, H⁺-translocating redox system. The electrochemical technique is a useful method of studying this system.     

Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.)

A new culture medium, originally designed and shown to grow cell suspensions from a variety of loblolly pine (Pinus taeda L.) explants, was used to study growth and somatic embryogenesis of the wild carrot (Daucus carota L.) in cell suspensions. The new loblolly pine medium (LM) differed from the standard wild carrot medium (WCM) in having very low Ca²⁺, very high Mg²⁺, and enrichment with PO _inf4 ^sup3– and microelements. When WCM was altered to contain levels of Ca²⁺ or Ca²⁺ and Mg²⁺ equivalent to LM, it supported neither growth nor embryogenesis of the wild carrot. However, growth and embryogenesis in LM was superior to WCM. The phosphate level in WCM was found to be suboptimal.     

Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by C NMR

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using ¹³C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.     

Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture

Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 × 10⁻⁴M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis. Received: 22 April 2000 / Accepted: 8 June 2000     

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.) : Evidence for Posttranslational Control of Calmodulin Expression

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 λgt10 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 [³⁵S]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.     

Investigations on the growth and metabolism of cultured explants of Daucus carota

Summary Earlier papers of this series relate to different growth-promoting substances and systems which, singly and in combination, have interacted with trace elements (Mn and Mo) and Fe to induce growth and to affect the metabolism of aseptic cultures of carrot. The solutes of cultured carrot cells (K⁺, Na⁺, Cl^–, total solutes) are also affected. Two clones were grown in 9 combinations of growth factors and under 4 trace-element regimes (a complete complement including Fe, and this complement lacking either Mn or Mo, or both Mn and Mo), a total of 36 treatments under otherwise standardized experimental conditions. Under the treatments applied the number of cells varied over a 35fold range and their average size over a 7fold range; the concomitant effects on their solutes are expressed in terms of concentrations and of total content per cell. Both growth and the solutes accumulated were variously affected by carrot growth-promoting system I (mediated by inositol), by system II (mediated by IAA), and by coconut milk in the presence of Fe, with and without Mn, Mo, or Mn and Mo.The greatest concentrations of total solutes occurred in tissue cultured in nutrient solutions which lacked the stimuli to rapid cell multiplication and were also limited by the trace elements Mn and Mo. Moreover, specific regulatory effects of the trace elements on solute content, not solely attributable to their effects on cell growth, have been noted. An imbalanced growth-factor regime (zeatin acting alone, i.e. without IAA) shifted the normal preference for K⁺ over Na⁺ strongly toward Na⁺, a trend which could also be induced by certain trace elements and more balanced growth-factor regimes, e.g. in a basal coconut milk medium lacking only Mn.The data are interpreted in the context of views on the de-novo uptake of salts and solutes in cultured cells as they grow. These cells respond to a network, or matrix, of interacting factors by distinctive effects that are attributable to the component parts of the culture medium acting singly and in various combinations. These interactions (involving trace elements and exogenous growth factors) control growth (fresh weight, number and size of cells) and regulate the solutes (organic and inorganic; K⁺ vs. Na⁺; organic anions vs. Cl^–) which the cells acquire as they grow and develop. The intensity of the response of the cultures to balanced, or imbalanced, growth factors creates the internal spaces accessible to solutes; and the metabolism, as it is also affected by growth factors and trace elements, determines how these spaces are to be filled at a given osmotic value. The evidence shows the range of factors that affect the accumulation of solutes in cells as they grow and is to be contrasted with conventional observations on mature cells held in steady states under conditions that preclude all growth and when only a single ionic species is followed over a very short interval of time.     

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2-³H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [³H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂). An additional [³H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP₂ on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.     

Characterization of a selenocystine-resistant carrot cell line : alterations in cystine and sulfate uptake

A selenocystine-resistant carrot cell line, C-1, was isolated from a haploid carrot (Daucus carota) cell culture, HA. The C-1 variant takes up cystine, but not cysteine, more slowly than does HA. The selenocystine resistance is maintained in culture in the absence of selection and is expressed in regenerated plants. Results based on chromatographic separation of sulfur metabolites from cells fed with [³⁵S]cystine suggest a block either in the uptake or reduction of cystine in the variant. Both lines can grow on cystine as sole sulfur source. Growth of the HA line on cystine suppressed the development of sulfate uptake capacity (Furner, Sung 1982 Proc Natl Acad Sci USA 79: 1149-1153), while cystine-grown C-1 cells have high levels of sulfate uptake capacity.

We suggest that the C-1 line, grown on cystine, accumulates an insufficient quantity of some sulfur metabolite, which is involved in the control of sulfate uptake, to suppress the uptake. C-1 grown on cystine is more sensitive than HA to growth inhibition by the sulfate analog selenate.

     

Detection,characterization, and phytotoxic activity of the nucleoside antibiotics,Blasticidin S and 5-Hydroxylmethyl-Blasticidin S

Screening for herbicidal compounds carried out on culture broths of Streptomyces strains isolated from soil resulted in the detection of potent phytotoxic activity. The active principles were isolated, and identified as the nucleoside antibiotics Blasticidin S (Bl-S) and 5-Hydroxymethyl-Blasticidin S (H-M-Bl-S). Bl-S was more active than H-M-Bl-S on seedling germination in petri dishes and in postemergence greenhouse tests. Moreover both antibiotics were more phytotoxic to dicotyledonous than to monocotyledonous plants. The increased sensitivity of dicots was confirmed in carrot and rice cell cultures. Both compounds also inhibited [¹⁴C]amino acid incorporation into proteins of rice and carrot cell cultures. Protein synthesis was more affected in carrot than rice.