Suspension Cultures Of Daucus Carota Research Articles

Effects of inoculum density, zeatin and sucrose on anthocyanin accumulation in a carrot suspension culture

Anthocyanin formation in a suspension culture of Daucus carota is induced by transfer from medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one lacking 2,4-D. The specific yields were strongly influenced by the inoculum density. Inoculum density altered the effect of zeatin concentration on anthocyanin accumulation. The in part by increasing the sucrose levels. It was inferred from the results that sucrose was exhausted at a low concentration of sucrose and at a high cell density, resulting in the decrease of yield of anthocyanin.

VARIATION OF PLASTID AND MITOCHONDRIAL DNAS IN THE GENUS DAUCUS

Plastid and mitochondrial DNAs from suspension cultures of Daucus carota subsp. sativus cv. Danvers, D. carota subsp. gummifer, D. capillifolius, and D. pusillus were compared by restriction endonuclease fragment analysis. Organelles isolated from protoplasts of suspension cultures were purified using a one‐step sucrose gradient. Plastid DNA fragment patterns for subsp. sativus, subsp. gummifer, and capillifolius were indistinguishable in Pstl, SalI and XhoI enzyme digests. No variation was detected in BamHI or HindIII digests between subsp. sativus and capillifolius. Pusillus plastid DNA varied significantly when compared to the other Daucus cultures. The size of the plastid genome of each species as determined by fragment addition, was estimated at 155 kb. Restriction digests of the mitochondrial DNAs generated a large number of fragments which when totaled established a size of 386–468 kb for the genomes. Densitometer scans of the fragment patterns indicate the bands were present in variable stoichiometry. Up to 26% of the fragments generated by PstI, SalI and HindIII digests were homologous in size among the four mitochondrial genomes. A further comparison of mitochondrial fragments indicated a closer homology of subsp. sativus to capillifolius than to subsp. gummifer or pusillus as was also found with the plastid genomes.

Variation of Plastid and Mitochondrial DNAs in the Genus Daucus

Plastid and mitochondrial DNAs from suspension cultures of Daucus carota subsp. sativus cv. Danvers, D. carota subsp. gummifer, D. capillifolius, and D. pusillus were compared by restriction endonuclease fragment analysis. Organelles isolated from protoplasts of suspension cultures were purified using a one-step sucrose gradient. Plastid DNA fragment patterns for subsp. sativus, subsp. gummifer, and capillifolius were indistinguishable in Pstl, SalI and XhoI enzyme digests. No variation was detected in BamHI or HindIII digests between subsp. sativus and capillifolius. Pusillus plastid DNA varied significantly when compared to the other Daucus cultures. The size of the plastid genome of each species as determined by fragment addition, was estimated at 155 kb. Restriction digests of the mitochondrial DNAs generated a large number of fragments which when totaled established a size of 386–468 kb for the genomes. Densitometer scans of the fragment patterns indicate the bands were present in variable stoichiometry. Up to 26% of the fragments generated by PstI, SalI and HindIII digests were homologous in size among the four mitochondrial genomes. A further comparison of mitochondrial fragments indicated a closer homology of subsp. sativus to capillifolius than to subsp. gummifer or pusillus as was also found with the plastid genomes.

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3'-position of ring B in Daucus carota takes place at the flavonoid stage.

Studies on the Regulatory Role of trans-Cinnamic Acid on the Activity of the Phenylalanine Ammonia-Lyase (PAL) in Suspension Cultures of Daucus carota L.

In vivo and in vitro experiments were performed in order to study the regulatory role of trans- cinnamic acid and its hydroxylated derivatives (p-coumaric acid, caffeic acid) on the deamina­tion of phenylalanine catalyzed by PAL (EC 4.3.1.5). Trans-cinnamic acid inhibits growth and reduces the content of soluble proteins of anthocyanin-containing carrot cells grown in suspen­sion. There is strong evidence from the polysomal patterns and from the effect of trans-cinnamic acid on protein synthesis in vitro that protein synthesis is inhibited. The kinetic data of PAL clearly demonstrate that trans-cinnamic acid inhibits the enzyme by a noncompetitive mecha­nism. On the contrary, ʟ-α-aminooxy-β-phenylpropionic acid (ʟ-AOPP), a competitive inhibitor of PAL, does not affect protein metabolism.

Selection and Characterization of Carrot Embryoid Cultures Resistant to Inhibition by Lysine Plus Threonine

Summary A lysine plus threonine resistant line was isolated by screening embryoid populations induced from a suspension culture of Daucus carota treated with N-methyl-N’-nitro-N-nitrosoguanidine.Amino add analysis showed that embryoids and callus of the resistant line contained at least 8 times more free threonine and 2.5 times more free isoleucine than the wild type.Lysine levels remained unchanged, methionine was hardly detectable both in wild type and resistant line tissues.The overproduction of threonine and isoleucine has been correlated with a decreased sensitivity to feedback inhibition of the lysine sensitive aspartate kinase.Resistant plants are cultivated further for genetical analysis.

Induction of mRNA activity for phenylalanine ammonia lyase (PAL) by L-α-aminooxy-β-phenylpropionic acid, a substrate analogue of L-phenylalanine, in cell suspension cultures of Daucus carota L

FEBS LettersVolume 146, Issue 1 p. 52-54 Full-length articleFree Access Induction of mRNA activity for phenylalanine ammonia lyase (PAL) by L-α-aminooxy-β-phenylpropionic acid, a substrate analogue of L-phenylalanine, in cell suspension cultures of Daucus carota L Wolfgang Noé, Wolfgang Noé Institut für Biologie I, Auf der Morgenstelle 1, 7400 Tübingen, FRGSearch for more papers by this authorHanns Ulrich Seitz, Hanns Ulrich Seitz Institut für Biologie I, Auf der Morgenstelle 1, 7400 Tübingen, FRGSearch for more papers by this author Wolfgang Noé, Wolfgang Noé Institut für Biologie I, Auf der Morgenstelle 1, 7400 Tübingen, FRGSearch for more papers by this authorHanns Ulrich Seitz, Hanns Ulrich Seitz Institut für Biologie I, Auf der Morgenstelle 1, 7400 Tübingen, FRGSearch for more papers by this author First published: September 06, 1982 https://doi.org/10.1016/0014-5793(82)80703-5Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume146, Issue1September 06, 1982Pages 52-54 ReferencesRelatedInformation

Induction of de novo synthesis of phenylalanine ammonia-lyase by L-?-aminooxy-?-phenylpropionic acid in suspension cultures ofDaucus carota L.

Application of L-α-aminooxy-β-phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283-287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.

Effect of medium constituents on the viability of immobilized plant cells

Cell suspension cultures of Daucus carota, Cannabis sativa, and Ipomoea sp. were entrapped within a matrix of calcium alginate. Immobilized D. carota cells were chosen as a model system for studies on the effects of medium constituents on the viability (i.e., respiration activity) of the entrapped cells. The 71 V medium could be simplified by omission of hormones, NH4+N, and vitamins, and 2-(N-morpholino)ethanesulphonic acid could substitute for phosphate as the buffering component of the medium. An examination of the effects of varying concentrations of sucrose, KCl, trace elements, and CaCl2 on the viability of the entrapped cells allowed a simple buffer mixture to be formulated. In this mixture, immobilized cells remained viable for at least 24 days and at 12 days could still transform digitoxigenin to periplogenin. Entrapped cells of C. sativa were similarly viable for at least 8 days.

Examination of parameters affecting the 5β-hydroxylation of digitoxigenin by immobilised cells of Daucus carota

Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl2, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl2 over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L-α-aminooxy-β-phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10(-4) M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10(-4) M) in the same sample. Carrot cells treated with L-AOPP exhibited a "super-induction" of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (K I=2.4·10(-9)), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (K I=1.8·10(-4)).

Expression of Aspartokinase, Dihydrodipicolinic Acid Synthase and Homoserine Dehydrogenase During Growth of Carrot Cell Suspension Cultures on Lysine-and Threonine-Supplemented Media

Reduction in the amounts of activity of the first enzyme, aspartokinase (EC 2.7.2.4) and two branch-point enzymes, dihydrodipicolinic acid synthase (EC 4.2.1.52) and homoserine dehydrogenase (EC 1.1.1.3), located in the pathway for the synthesis of aspartate-family amino acids, occurred when cell suspension cultures of Daucus carota L. var. Danvers were grown in media containing 2 mM threonine or 2 mM lysine, endproducts of the pathway. Activity of the lysine-sensitive form of aspartokinase was decreased when cells were grown in medium containing lysine and the activity of the threonine-sensitive form was decreased when cells were grown in medium containing threonine. Activity of the branch-point enzyme leading to threonine synthesis, homoserine dehydrogenase, was decreased up to 70% in specific activity (units/mg protein) and relative activity (units/g fresh weight) when cells were grown in media containing lysine or threonine. Threonine had no effect on the relative activity of dihydrodipicolinic acid synthase, but decreased its specific activity. Lysine decreased the relative activity of the synthase by up to 40%, but had little effect on its specific activity. The decreased activities of the enzymes were apparently not due to binding of the inhibitory amino acids to the enzymes since homogenization of cells in buffer with 2 mM lysine and threonine did not decrease the measurable enzyme activities. These and other results presented suggest that both forms of the aspartokinase activity and homoserine dehydrogenase activity can be altered by supplementing the growth medium with lysine or threonine.

Isolation and Characterization of Vacuoles from Cell Suspension Cultures of Daucus carota

Abstract A reliable procedure for the isolation of vacuoles from anthocyanin-containing cells of Daucus carota cell suspension cultures has been developed. From cells of the late linear growth phase, protoplasts were prepared and purified in a sucrose/sorbitol gradient. Vacuoles were liberated from these protoplasts by osmotic shock and purified in a Metrizamide step gradient. The vacuolecontaining fractions were analysed for their anthocyanin content as a measure for the yield of vacuoles. The purity of vacuoles was examined by assaying various marker enzymes in both protoplasts and vacuoles. The purest vacuolar fraction had 8% of the total activity of glucose-6-phosphate dehydrogenase (marker for cytosol), 8% of cytochrome oxidase (mitochondria) and 10% of NADPH -cytochrome c reductase (ER). 55% of the acid phosphatase activity of the protoplasts and 35% of the total malate were recovered in the vacuoles. The vacuolar pool of amino acids is quite large. Data for 15 amino acids show that 44 to 73% are being located in the vacuole.

Oleosomes (Spherosomes) from Daucus carota suspension culture cells

Isolated oleosomes from Daucus carota L. cells are lipid droplets consisting mainly of triacylglycerols (>97%) and very little protein (1-2%). The boundary between the lipid phase and the cytosol, which is visible on electron micrographs, is not built up by a true phospholipid-containing unit or half unit membrane. Enzymatic activities of lipid metabolism were not found to be associated with oleosomes with the exception of very low (contaminating) acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) and relatively high acyl-CoA hydrolase (EC 3.1.2.2) activities. The triacylglycerols exhibited a half life time of about 70 h, which is below the generation time of the cells (80-90 h). The fatty acid pattern of triacylglycerols was very similar to that of polar cellular membrane lipids.

Factors affecting the frequency of tetraploid cells in a predominantly diploid suspension culture ofDaucus carota

Tetraploid clones were isolated from a predominantly diploid culture line ofDaucus carota by plating alone or after colchicine treatment. Although individually these tetraploid clones had similar growth rates to the diploid culture, they were progressively eliminated from mixtures with the diploid line. As the diploid culture line always contained a low frequency of tetraploids, the selective elimination of tetraploids must have been balanced by their continuous production. The frequency of endoreduplication detected was too low to maintain the equilibrium frequency of tetraploids and it is proposed that polyploidisation also occurred by endomitosis. The mean frequency of tetraploid metaphases within the diploid culture line was reduced by shortening the interval between subcultures from 14 to 7 days. This 7-day regime also eliminated linear growth and stationary phase periods, but did not alter the maximum growth rate or mitotic index of the culture. It is argued that the change in mean tetraploid metaphase frequency is indicative of a change in the number of tetraploid cells within the culture, brought about by an alteration in the equilibrium between production and elimination of tetraploid cells.

The effects of 2,4-D on growth and mitosis in suspension cultures of Daucus carota

The mean generation time of suspension cultures of Daucus carota was linearly related to 2,4-dichlorophenoxyacetic acid (2,4-D) concentration over the range 0.1–20 μg ml −1. Cell cycle analysis indicated that suppression of growth by 2,4-D was due to an extension of, or blockage in the G1 or G2 stages of the cell cycle and was associated with lignin deposition at concentrations above 15 μg ml −1. At 2,4-D concentrations from 0.1–30 μg ml −1, mitotic duration increased progressively and mitotic stage proportions were altered such that the durations of prophase and metaphase were increased relative to those of anaphase and telophase. This suggested that 2,4-D was lengthening the period during which the mitotic spindle was assembled and might prevent spindle formation at concentrations greater than 30 μg ml −1.

Morphometric measurements of Daucus carota suspension culture cells

Standard stereologic methods have been applied to electron micrographs of Daucus carota L. suspension culture cells. The relative frequencies of the different membrane systems within the cells have been determined and compared with published data form mature leaf cells.

Uptake of 14C‐L‐Glutamic Acid by Daucus carota Cell Suspension in Different Shock Situations

AbstractCell suspension cultures of Daucus carota were shocked by inoculation or by temperature, salts or osmotica in combination with inoculation. Changes in cell vitality were measured by uptake studies with labelled L‐glutamic acid performed during the first 30 min after shock treatment. The cell sample was then collected on filter and counted in a liquid scintillation counter. Inoculation alone caused a marked decrease in uptake. Normal uptake capacity was re‐established within 2 h. Temperatures of 0−20°C as against 27°C gave an additional decrease in uptake. Of the tested osmotica, polyethylene glycol gave the greatest stabilizing effect on the glutamic acid uptake and sucrose the least. The highest uptake rates were obtained with a calcium concentration of 0.10–0.15 mM.

Variation of cell cycle duration within suspension cultures of Daucus carota and its consequence for the induction of ploidy changes with colchicine.

Continuous exposure to colchicine was used to estimate the variation in cell cycle time between cells within suspension cultures ofDaucus carota. Observations were made of the pattern of disappearance of cells of the initially predominant ploidy levels in diploid and tetraploid cultures having markedly different aggregation patterns. Both cultures showed a similar range of cycle times, normally distributed about the culture mean. Shorter colchicine treatments, followed by regrowth in colchicine-free medium, showed that spread of cycle times in the diploid culture prevented uniform induction of tetraploidy, and that the resulting mixoploid suspensions showed a gradual reversion to diploidy during subsequent subculture.

Protoplast Cultures from Cell Suspensions of Daucus carota

AbstractProtoplasts, enzymatically isolated from cell suspension cultures of Daucus carota, have been grown in small Petri‐dishes. After enzyme treatment and washing the protoplasts were plated in agar media. Growth and divisions were viewed through the bottom of the Petri‐dishes with a light microscope. Different osmotic stabilizers were tested with respect to their ability to promote wall formation and growth of the protoplasts. Combinations of sucrose, sorbitol and “Modopeg” gave the best results. Electron micrographs of cultured protoplasts revealed normal as well as abnormal nuclear conditions.