Cultured Carrot Cells Research Articles

Effect of anti-microtubular drug amiprophos-methyl on somatic embryogenesis and DNA ploidy levels in alfalfa and carrot cell suspension cultures

A short treatment with the anti-microtubular drug amiprophos-methyl (APM) blocked somatic embryogenesis in alfalfa(Medicago sativa L.) and carrot (Daucus carota L.). The interruption was temporary and restoration of somatic embryogenesis was observed in long-term cultures. In addition to the effect on somatic embryogenesis, APM treatment induced polyploidization the extent of which was concentration dependent. In long-term alfalfa cultures, APM-induced loss of somatic embryogenesis led to ploidy instability and to a shift to DNA aneuploidy. Critical stages of somatic embryogenesis sensitive to disruption of microtubule-mediated processes were determined in carrot cell cultures. Complete embryogenic arrest occurred when APM was added within the first 5 d of embryogenesis from single cells. The role of the cytoskeleton in the first events of somatic embryogenesis and the relation between totipotency and ploidy stabilityin vitro is discussed.

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.

Synthesis and Degradation of Cyclic AMP in Cultured Carrot Cells Treated with Forskolin

Synthesis and Degradation of Cyclic AMP in Cultured Carrot Cells Treated with Forskolin

Loss of tubulin during cold treatment of cultured carrot cells

Loss of tubulin during cold treatment of cultured carrot cells

Loss of tubulin during cold treatment of cultured carrot cells

When carrot cells, Daucus carota L. cv. Kintoki, in suspension culture were chilled on ice, more than 90% of the cortical microtubules disappeared within an hour. Colchicine‐binding activity in the soluble extract decreased gradually during the prolonged cold treatment and after 4 days reached a minimum level. In SDS‐polyacrylamide gel electrophoretograms of the cold‐treated cell extracts, the bands corresponding to tubulin subunits disappeared, whereas most of the other protein bands remained. The level of tubulin was restored within a day after the cells were returned to 267deg;C. The relative ratio of the two major β‐tubulin isoforms was reversed during the cold treatment, indicating that the cold sensitivity of tubulin differs according to its molecular species. When the cells were returned to 26°C, the ratio was restored to the original state within 24 h.

Stimulation of Calcium Influx and Calcium Cascade by Cyclic AMP in Cultured Carrot Cells

Treatment of cultured carrot (Daucus carota L.) cells with activators of adenylate cyclase, forskolin, and cholera toxin induced the biosynthesis of an antifungal isocoumarin, 6-methoxymellein, in the cells. Addition of dihutyryl cyclic AMP to carrot cell culture also stimulated the accumulation of the compound. The cyclic AMP-evoked 6-methoxymellein production was significantly depressed in the presence of certain inhibitors of calcium cascade such as Ca2+ channel blockers and inhibitors of calmodulin-dependent processes. In dibutyryl cyclic AMP- and forskolin-treated carrot cells, increase in cytosolic Ca2+ concentration was observed as monitored by the fluorescent calcium indicator fluo-3. Cyclic AMP-dependent Ca2+ influx into carrot cells was also confirmed with Ca2+-loaded vesicles prepared from the plasma membrane-rich fraction of the cells. Transient increase in Ca2+- and Ca2+/calmodulin-dependent protein kinase activity but not cyclic AMP-dependent protein phosphorylation was detected in the cells of high cyclic AMP concentration. Results obtained in the present work suggest that the increase in cyclic AMP content in carrot cells induces Ca2+ influx across plasma membrane without activating cyclic AMP-dependent protein kinase which, then, stimulates calcium cascade in the cells.

A Herbicidal Inhibitor of Isopropylmalate Isomerase

Nitronate compounds resembling the aci-carboxylate reaction intermediate in the reaction catalyzed by isopropylmalate isomerase were investigated as enzyme inhibitors and potential herbicides. The nitronic acids of 1-hydroxy-2-nitrocyclopentane-1-carboxylic acid, nitroisopropylmalate and of 1-hydroxy -2-nitrocyclohexane-1-carboxylic acid were all potent inhibitors of isopropylmalate isomerase. The nitroalkane/nitronate pK a values of these were 7.3, 9.5 and 11.1, respectively. The perturbed pK a values of the first and last compound were und erstandable on the basis of the opposing effects of 5- and 6-membered ring strain on the ionization of the nitro group. As the only compound significantly in the nitronate form at physiological pH , it was not surprising that 1-hydroxy-2-nitrocyclopentane-1-carboxylic acid was the best herbicide. Its specific mode of action was confirmed by the fact that 1-hydroxy-2-nitrocyclopentane- 1-carboxylic acid inhibited the growth of carrot cells in liquid culture and was specifically reversed by leucine.

Enhancement and repression of somatic embryogenesis in cell cultures of carrot by cold pretreatment of stock plants

Roots of nine cultivars of carrot (Daucus carota L.) were exposed to one, two or three months cold treatment and cells isolated from cold-treated and control roots were assayed for the production of somatic embryos. Cells obtained from the one- or two-months cold treatments formed embryos earlier, produced embryos over a longer period of time and produced more embryos per callus than the controls. In contrast, cells obtained from roots exposed to three months cold displayed a reduction in all parameters of embryogenic competence and for some cultivars this treatment resulted in production of cells with no embryogenic competence. The relationship of cold treatment of stock plants to the induction of metastable and stable patterns of gene expression and the induction of somatic embryos are discussed.

Changes in vacuolar pH of carrot cells in suspension culture grown under saline conditions

The effect of salinity on vacuolar pH was studied in carrot (Daucus carota L.) cells grown in liquid suspension culture either in the absence or presence of 150 mM NaCl. Both vacuolar and cytoplasmic pH were determined by several independent techniques. These techniques were NMR spectrometry, distribution of radioactive probes and spectrophotometric measurement of the absorbance changes of a naturally occurring vacuolar pH indicator. There was no difference in the cytoplasmic pH between cells grown in the presence or the absence of NaCl, but the vacuolar pH of cells grown in the presence of NaCl was higher by 0.38 to 1.05 pH units (depending on the technique that was used) than the vacuolar pH of cells grown in the absence of NaCl.

Differential accumulation of storage products in developing seeds and somatic cell cultures of Daucus carota L.

The major oleosin proteins of 15 and 19 kDa and the major storage albumins of 20 and 28 kDa were purified to homogeneity from carrot seeds and antibodies raised against them. Anti-oleosin antibodies recognised similar sized proteins in total protein and oil body fractions from non-embryogenic and embryogenic carrot suspension cultures and from carrot somatic embryos. Both oleosins and oil bodies accumulated in all carrot cell and tissue types but reached much higher levels in high osmoticum (0.4 M sucrose or sorbitol) than in basal media. In contrast, there was no evidence of storage protein accumulation in any carrot cell or tissue cultures in vitro, even in the presence of high osmoticum or plant growth regulators. The carrot cultures in vitro did not accumulate the high levels of octadecenoic fatty acids found in seeds but rather accumulated an oil which was high in linoleic acid and whose fatty acid profile resembled that of the membrane lipids. Non-embryogenic carrot cell cultures accumulated very little starch, whereas high levels were found in embryogenic cell cultures. Starch levels declined to low levels during somatic embryo development to the torpedo stage. The differences in the regulation of accumulation of oleosins, storage albumins, triacylglycerols and starch in vitro and in planta in carrot cells and tissues are discussed.

Characterization of the glyphosate selection of carrot suspension cultures resulting in gene amplification

When Daucus carota L. (carrot) suspension cultures, which had been under gradual selection for increased glyphosate resistance, whre plated in glyphosate-containing medium, the populations became gradually more resistant with I 50 values about equal to the final selection concentration used in the liquid medium. Wild type cells did not grow on plates with 2 mM glyphosate, but some colonies formed after 90 days. Cells in these colonies, after growth away for the inhibitor to obtain sufficient cells, had increased glyphosate resistance and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity and usually had amplified EPSPS genes. One step selection in liquid or solidified medium with 10 mM glyphosate was not successful. However, in liquid medium with 2 mM glyphosate, the cell viability decreased to about 30% by 30 days and then increased as growth became evident. Kinetic analysis indicates that at least 0.8% of the cells grow. The growth rate seen when the cells are subjected to a gradual selection scheme, where glyphosate was about doubled at each step, also indicates that a high proportion of the original population survives to form the new population. These results indicate that the gene amplification which causes the glyphosate tolerance in carrot cell cultures is a gradual and relatively stable process that occurs in a relatively high proportion of the cell population.

Plant chitinases

Plant chitinases

Regulation of Indole-3-Acetic Acid Biosynthetic Pathways in Carrot Cell Cultures

2,4-Dichlorophenoxyacetic acid (2,4-D) promotes the accumulation of tryptophan-derived indole-3-acetic acid (IAA) in carrot cell cultures during callus proliferation by a biosynthetic pathway that is apparently not active during somatic embryo formation. The effects of 2,4-D were examined by measuring the isotopic enrichment of IAA due to the incorporation of stable isotope-labeled precursors (deuterium oxide, [(15)N]indole, and (2)H(5)-l-tryptophan). Enrichment of IAA from deuterium oxide is similar in both cultured hypocotyls and cell suspension cultures in the presence and absence of 2,4-D, despite the large differences in absolute IAA concentrations. The enrichment of IAA due to the incorporation of [(15)N]indole is also similar in callus proliferating in the presence of 2,4-D and in embryos developing in the absence of 2,4-D. The incorporation of (2)H(5)-l-tryptophan into IAA, however, is at least 7-fold higher in carrot callus cultures proliferating in the presence of 2,4-D than in embryos developing in the absence of 2,4-D. Other experiments demonstrated that this differential incorporation of (2)H(5)-l-tryptophan into IAA does not result from differential tryptophan uptake or its subsequent compartmentation. Thus, it appears that differential pathways for IAA synthesis operate in callus cultures and in developing embryos, which may suggest that a relationship exists between the route of IAA biosynthesis and development.

Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures

A bioreactor control system was used to investigate the effects of two dissolved oxygen concentrations (10% and 100%) on the growth and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled without the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry biomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation was highly sensitive to reduced oxygen since under this condition, the somatic embryo production was inhibited by about 75%. Sugar uptake and embryo formation were also delayed.

Single-stranded DNA of 5′-upstream region of the rolC gene interacts with nuclear proteins of carrot cell cultures

Using the gel retardation assay, proteins of carrot cells capable of binding to a single-stranded DNA of 5′-upstream region of the rolC gene were found. From competition experiments, these DNA-protein interactions were specific to single-stranded nucleotide sequence of Ava S fragment (from ∝-94 by to +23 by relative to the transcription initiation site). Methylation interference experiments showed that G residue at the position of −41 bases on the bottom strand was important for DNA-protein binding. This residue was located between CART box and TATAA box. Such specific interaction between single-stranded DNA and nuclear proteins may play a role in transcription by RNA polymeraw II.

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/105 cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.

Modulation of calmodulin levels, calmodulin methylation, and calmodulin binding proteins during carrot cell growth and embryogenesis

Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M r calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development.

Ploidy reduction and genome segregation in cultured carrot cell lines. II. Somatic meiosis

Carrot cell lines W1 and W2 express permanently in culture a meiotic-like phenotpe, with apparent pairing and chiasmata formation comparable to meiosis during carrot microsporogenesis. The variant lines also show several variants of division in relation to the presence or absence of cytokinesis, nuclear fusion or spindle disturbance. The meiotic-like divisions can also be found in the abnormal structures, which are regenerated from these spontaneous variant lines. A possible role of the chromosome reducing mechanisms on carrot embryogenesis capacity and somaclonal variability is postulated.

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner. The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.

An extracellular β‐galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall‐polysaccharide hydrolysis

β‐Galactosidase (β‐Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot (Daucus carota L. cv. Kintoki). The extracellular β‐Galase (β‐Galase‐II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM‐Sephadex C‐50. DEAE‐Sepharose CL‐6B and Sephacryl S‐200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S‐200HR gel‐permeation, and 60 kDa by SDS‐PAGE after treatment with SDS and 2‐mercaptoethanol. The pI was 6.5. The Km and Vmax values for p‐nitrophenyl (PNP)‐β‐D‐galactopyranoside were 0.17 mM and 31.9 μmol (mg protein)‐1, h‐1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co24, Cu2+, Hg2‐, p‐chloromercuribenzoate (PCMB) and D‐galactono‐1,4‐lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo‐fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.