Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

The effects of plant growth regulators were investigated onanthocyanin synthesis induced by removing auxin from carrotsuspension cultures. Of the auxins tested, 2,4-D showed thestrongest inhibiting effect on anthocyanin synthesis and hadthe strongest promoting effect on undifferentiated growth. When2,4-D was added to anthocyanin synthesizing cells, in whichcell division had ceased, anthocyanin synthesis was repressedimmediately, accumulated anthocyanin disappeared and cell divisionresumed. All cytokinins examined promoted anthocyanin synthesisin the absence of auxin. Both gibberellic acid (GA₃) and abscisicacid inhibited anthocyanin synthesis in media lacking 2,4-D,though GA³ showed no effect on cell division. These effectsof growth regulators on anthocyanin synthesis are similar tothose reported for their effects on embryogenesis [Fujimuraand Komamine (1975) Plant Sci. Lett. 5: 359, (1979) Z. Pflanzenphysiol.95: 13, (1980) Z. PJlanzenphysiol. 99: 1]. The relationshipbetween the induction of anthocyanin synthesis, metabolic differentiation,and embryogenesis are discussed. ¹ Present address: Department of Biology, College of Arts andSciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo153, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted July 23, 1986)     

The Origin and Development of Embryoids in Suspension Cultures of Carrot (Daucus carota)

In suspension cultures of carrot, embryoids are initiated ator near the surface of characteristic cellular aggregates (embryogenicclumps) and are released from these aggregates as free-floatingstructures capable of further development into plantlets. Embryoiddevelopment is promoted by transfer from a medium containing2,4-D to one from which it is omitted. The structure of theembryogenic clumps, in these two media, has been studied bythe thin section technique involving fixation in glutaraldehydeand embedding in glycol methacrylate. The superficial cellsof the clumps are clearly marked off from the central cellsand contain large starch grains, a large central nucleus, abundantcytoplasm and a number of vacuoles. It is from individual superficialcells that the embryoids are seen to arise in the 2,4-D omittedmedium but it has not been possible to distinguish, in advanceof embryogeny, such cells from within the superficial cell layers.The fragmentation of the proliferating clumps in 2,4-D containingmedium and the release of embryoids into the 2,4-D omitted mediumis promoted by some of the superficial cells becoming free ofstarch and undergoing pronounced enlargement. A sequence of segmentations leading from single superficialcells of the clumps to globular embryoids has been traced. Thisdiffers from that reported to occur during early embryogenyfrom the egg cell of carrot. The resemblance to zygote embryogenyis closer from the late globular stage of embryoid development;embryoids then differ from zygotic embryos in their shortersuspensors. Attention is drawn to the initiation of additionalembryoids from the epidermis of some of the embryoids developingin culture.     

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.     

Dynamics of Nitrogenous Assimilate Partitioning between Cytoplasmic and Vacuolar Fractions in Carrot Cell Suspension Cultures

Bulk vacuole isolation, gas chromatography-mass spectrometry,, and high-performance liquid chromatography have been used to investigate the accumulation and partitioning of assimilated nitrogen supplied as ¹⁵NH₄Cl between vacuolar and extravacuolar (cytoplasmic) fractions of protoplasts from suspension cultures of carrot (Daucus carota L. cv Chantenay). Glutamine was the most abundant amino acid in the vacuole of protoplasts from late-exponential phase cells, whereas alanine, glutamate, and γ-aminobutyric acid were located primarily in the cytoplasmic fraction. In ¹⁵N-feeding studies, newly synthesized glutamine partitioned strongly to the vacuole, whereas glutamate partitioned strongly to the cytoplasm, γ-aminobutyric acid was totally excluded from the vacuole, and alanine was distributed in both compartments. Comparison of the ¹⁵N-enrichment patterns suggests that initial assimilation to glutamine occurs within a subcompartment of the cytoplasmic fraction. The protoplast-feeding technique may be extended to investigate cytoplasmic compartmentation further.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

Quantitative Measurement of the Diastereoisomers of CIS Thymidine Glycol in Gamma-Irradiated DNA

A technique for determing the relative content of each of the diastereoisomers of cis thymidine glycol (dTG) in DNA exposed to ionizing radiation has been developed. [³H]thymidine DNA was gamma-irradiated, digested to 2′-deoxyribonucleosides, authentic [¹⁴C] (+, -) cis dTG added to the digestate and the mixture resolved by HPLC. ³H fractions coeluting with [¹⁴C] (+, -) dTG were collected and acetylated.

The acetoxy derivatives.of (+) and (-) cis dTG were easily resolved by a second HPLC analysis and their absolute configuration determined by NMR amd mass spectroscopies. We have constructed a dose-response curve for formation of each isomer in gamma-irradiated DNA and shown that they are formed in equal amounts. This technique may be used to determine the relative formation of cis dTG isomers in DNA resulting from other oxidative stresses and whether repair of these is influenced by their configuration.     

Quantitative Measurement of the Diastereoisomers of CIS Thymidine Glycol in Gamma-Irradiated DNA

A technique for determing the relative content of each of the diastereoisomers of cis thymidine glycol (dTG) in DNA exposed to ionizing radiation has been developed. [³H]thymidine DNA was gamma-irradiated, digested to 2'-deoxyribonucleosides, authentic [¹⁴C] (+, -) cis dTG added to the digestate and the mixture resolved by HPLC. ³H fractions coeluting with [¹⁴C] (+, -) dTG were collected and acetylated.

The acetoxy derivatives.of (+) and (-) cis dTG were easily resolved by a second HPLC analysis and their absolute configuration determined by NMR amd mass spectroscopies. We have constructed a dose-response curve for formation of each isomer in gamma-irradiated DNA and shown that they are formed in equal amounts. This technique may be used to determine the relative formation of cis dTG isomers in DNA resulting from other oxidative stresses and whether repair of these is influenced by their configuration.     

Determination of Specific Growth Rate by Measurement of Specific Rate of Ribosome Synthesis in Growing and Nongrowing Cultures of Acinetobacter calcoaceticus

RT-RiboSyn measures the specific rate of ribosome synthesis in distinct microbial populations by measuring the generation rate of precursor 16S rRNA relative to that of mature 16S rRNA when precursor 16S rRNA processing is inhibited. Good agreement was demonstrated between specific rate of ribosome synthesis and specific growth rate of Acinetobacter calcoaceticus.     

Synthesis and Transport of Hydroxyproline-rich Components in Suspension Cultures of Sycamore-Maple Cells

Plant cell walls contain a glycoprotein rich in hydroxyproline. To determine how Acer pseudoplatanus L. cells transport this glycoprotein to the wall, the pulse-chase technique was used to follow changes in specific radio-activity of hydroxyproline and proline in isolated, mitochondrial, Golgi, microsomal, soluble protein, and wall fractions. The turnover rates or changes in specific radioactivity of cytoplasmic hydroxyproline in these cell fractions indicated that the bulk of this hydroxyproline was transferred not by the Golgi apparatus but by a smooth membranous component.     

Regulation of Glutathione Synthesis in Suspension Cultures of Parsley and Tobacco*

Experiments in vitro have shown that γ-EC synthesis, the first step in GSH formation, is subject to feedback inhibition by physiological GSH concentrations. In order to evaluate the role of this feedback inhibition on γ-EC synthetase in vivo GSH synthesis was modulated in suspension cultures of P. crispum and N. tabacum by administration of cadmium. The alterations in the thiol contents were measured and in addition the effect of Cd exposure on γ-EC synthetase (E.C. 6.3.2.2) and GSH synthetase (E.C. 6.3.2.3) was studied. Decreasing cellular GSH concentrations by cadmium induced PC synthesis caused 7–10 fold increase in the rate of glutathione synthesis as measured by the accumulation of (γ-EC)_nG. This increase was not linked to an increase in extractable activities of γ-EC- or GSH synthetase in parsley. In tobacco the activities of γ-EC- and GSH synthetase increased by a factor of 1.6 and 1.8, respectively, after 3 d of Cd exposure. In both species the exposure to Cd resulted in an increased cellular γ-EC content that reached a plateau within 24 h, and in a doubling of the cysteine content. In vitro experiments showed that GSH synthetase activity is inhibited by cadmium concentrations that have no effect on γ-EC synthetase activity. This may explain the accumulation of γ-EC in Cd exposed cells. Incubation with 0.25 mM cysteine did not effect the γ-EC- and GSH content in tobacco cells. In parsley the cellular GSH content increased threefold and the y-EC content twofold and stayed constant thereafter at the elevated levels. Taken together the results show that GSH synthesis in vivo is controlled by feedback inhibition as well as by the supply with cysteine. In the latter case the feedback inhibition may act as a kind of safety valve and prevent the accumulation of unphysiological GSH concentrations if the supply of cysteine is too large.     

Synthesis and Turnover of Cell-Wall Polysaccharides and Starch in Photosynthetic Soybean Suspension Cultures

Soybean (Glycine max [L.] Merr.) suspension cultures grown under photoautotrophic and photomixotrophic (1% sucrose) culture conditions were used in 14CO2 pulse-chase experiments to follow cell-wall polysaccharide and starch biosynthesis and turnover. Following a 30-min pulse with 14CO2, about one-fourth of the 14C of the photoautotrophic cells was incorporated into the cell wall; this increased to about 80% during a 96-h chase in unlabeled CO2. Cells early in the cell culture cycle (3 d) incorporated more 14C per sample and also exhibited greater turnover of the pectin and hemicellulose fractions as shown by loss of 14C during the 96-h chase than did 10- and 16-d cells. When the chase occurred in the dark, less 14C was incorporated into the cell wall because of the cessation of growth and higher respiratory loss. The dark effect was much less pronounced with the photomixotrophic cells. Even though the cell starch levels were much lower than in leaves, high 14C incorporation was found during the pulse, especially in older cells. The label was largely lost during the chase, indicating that starch is involved in the short-term storage of photosynthate. Thus, these easily labeled and manipulated photosynthetic cells demonstrated extensive turnover of the cell-wall pectin and hemicellulose fractions and starch during the normal growth process.     

Light-Induced Synthesis of Anthocyanin in Carrot Cells in Suspension: I. THE FACTORS AFFECTING ANTHOCYANIN PRODUCTION

Takeda, J. 1988. Light-induced synthesis of anthocyanin in carrotcells in suspension. I. The factors affecting anthocyanin production.—J.exp. Bot. 39: 1065–1077. A light-triggered anthocyanin-synthesizing system was establishedfor carrot cells in suspension. A few days after transfer ofthe cells to a 2,4-dichlorophenoxyacetic acid (2,4-D)-free mediumin the dark, light irradiation triggered anthocyanin synthesisand concomitantly stopped expansion growth. Over 90% of thecells synthesized anthocyanin without cell division. By loweringthe concentration of phosphate or both nitrogen and phosphateand delaying the time of onset of irradiation, the productionof anthocyanin per cell increased to a maximum level of 0–8µmol anthocyanin per 10⁶ cells. A change in the physiologicalstate of cells (light-insensitive to light-sensitive state)induced by the transfer to 2,4-D-free medium is suggested tobe a prerequisite for the light-triggered synthesis of anthocyanin. Key words: Anthocyanin production, cultured cells, Daucus carota, light-triggered, 2,4-D     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

Changes in Activities of Enzymes Involved in General Phenylpropanoid Metabolism during the Induction and Reduction of Anthocyanin Synthesis in a Carrot Suspension Culture as Regulated by 2,4-D

The activities of enzymes involved in general phenylpropanoidmetabolism were followed in a carrot suspension culture duringthe induction and reduction of anthocyanin synthesis regulatedby 2,4-D. When no anthocyanin synthesis occurred in a mediumcontaining 2,4-D (+2,4-D medium), the activities of phenylalanineammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) increased1 day after transfer due to the transfer effect, but subsequentlydecreased and remained at a low level. Cinnamate-4-hydroxylase(C4H) activity showed a low level throughout culture. When cellswere transferred to a medium lacking 2,4-D (–2,4-D medium),the activities of PAL, C4H and 4CL increased and maximum activitiesof these enzymes were observed 6–7 days after transfer,when anthocyanin was most rapidly synthesized. When cells were cultured in the –2,4-D medium, the additionof 2,4-D immediately reduced the induced activity of PAL. PALactivity was super-induced by the transfer effect, while anthocyaninsynthesis decreased. The addition of intermediates of generalphenylpropanoid metabolism, with 2,4-D, to the medium 6 daysafter transfer to the –2,4-D medium did not promote anthocyaninsynthesis, whereas dihydroquercetin did promote it. Regulationof anthocyanin synthesis by 2,4-D is discussed in relation tochanges in enzyme activities involved in general phenylpropanoidmetabolism. ¹ Present address: Cell Science and Technology Division, FermentationResearch Institute, Agency of Industrial Science and Technology,Yatabe-machi, Ibaraki 305, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : IV. Induction of Vanillic Acid Formation

Kinetin is used as an elicitor to induce vanillic acid formation in cell suspension cultures of Vanilla planifolia. Maximal induction is observed at a kinetin concentration of 20 micrograms per gram of fresh weight of cells. Vanillic acid synthesis is observed a few hours after elicitation. The effects of kinetin on the activity of some enzymes of the phenylpropanoid pathway, i.e. phenylalanine ammonia-lyase, 4-hydroxycinnamate:coenzyme A ligase and uridine 5′-diphosphate-glucose:trans-cinnamic acid glucosyltransferase, are reported and compared to the effects of chitosan. The former two enzymes are induced by chitosan with a maximum activity of approximately 25 to 40 hours after elicitation. All three enzymes are induced by kinetin with maximum activities for phenylalanine ammonia lyase and 4-hydroxycinnamate:coenzyme A ligase at approximately 50 hours after induction, whereas maximum glucosyltransferase activity is seen already after 24 hours. Furthermore, both elicitors induced the formation of lignin-like material, whereas only kinetin induced vanillic acid biosynthesis. Finally, kinetin but not chitosan induces catechol-4-O-methyltransferase activity, catalyzing the formation of 4-methoxycinnamic acids, which were shown to be intermediates of hydroxybenzoic acid biosynthesis within cells of V. planifolia. It is suggested that this methyltransferase is directly involved in the biosynthesis of vanillic acid.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Nitrogen Metabolism in Plant Cell Suspension Cultures: II. Role of Organic Acids during Growth on Ammonia

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from ¹⁴C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes.     

Effects of Kinetin on Growth and Nucleic Acid Metabolism in Suspension Cultures of L. Cucumis melo

At 0.05 and 0.5 mg per 1 of kinetin growth of cucumber suspensioncultures were promoted and at a higher level (5.0 mg per 1)it was inhibited. Total nucleic acids and protein increasedduring the lag phase and declined during the phase of exponentialgrowth. The nucleotides increased before there was any appreciableincrease in the amount of supernatant ribonucleic acid. TheRNA, soluble protein and ribo-nuclease activity also rose duringthe early period of growth and subsequently fell. Higher amountsof nucleotides, RNA and soluble protein were present at growthpromoting kinetin concentrations, but these were in smalleramounts at supra-optimal kinetin levels. But the rate of declineduring the period of increase in dry weight was slower comparedto the rest. There was reduced activity of RNase, which alsofollowed RNA and soluble protein in the pattern of changes duringthe course of culture.