Daucus Carota Cell Research Articles

ENANTIOSELECTIVE SYNTHESIS OF (S)-(-)-1-(4-FLUOROPHENYL)ETHANOL

S)-(–)-1-(4-Fluorophenyl)ethanolis an intermediate in the synthesis of an antagonist of the CCR5 chemokine receptor that can be protective against HIV infection. Along with o-fluoro-substituted analogs, (S)-(–)-1-(4-fluorophenyl)ethanol can be used to study the role of substituents in the chiral recognition of molecular complexes, and (R)-(+)-1-(4-fluorophenyl)ethanol is a com-ponent of an antimalarial drug and a γ-secretase modulator necessary for the treatment of Alzhei-mer's disease.We proposed an approach to the synthesis of (S)-(-)-1-(4-fluorophenyl)ethanol based on the enantioselective reduction of prochiral 1-(4-fluorophenyl)ethanone catalyzed by Daucus carota cells. We found the conditions that make it possible to obtain (S)-(–)-1-(4-fluorophenyl)eth-anol by bioreduction of 1-(4-fluorophenyl)ethanone in the presence of a D. carota biocatalyst for 48 h with 55% yield (98% ee).We also studied the possibility of optimizing the transformation of 1-(4-fluorophenyl)ethanone in a reaction medium containing various exogenous reducing agents(ethanol, isopropanol, or glucose), which are known to significantly affect the product yield and the enantioselectivity of the process. Biotransformation of 1-(4-fluorophenyl)ethanone in the pres-ence of ethanol (1-5%) gives (S)-(-)-1-(4-fluorophenyl)ethanol with 7-85% yields and optical purity 10-98% ee.Enantioselective reduction of the initial ketone in the presence of isopropanol (1-5%) does not lead to a significant increase in the yield and optical purity of S-alcohol (7-57% (18-98% ee).In the case of biotransformation of 1-(4-fluorophenyl)ethanone in the presence of an equimo-lar amount of glucosefor 48 hthe yield of (S)-(-)-1-(4-fluorophenyl)ethanol is 66% (98% ee).Fur-ther transformation of the initial ketone (144 h) both in the absence of an exogenous reducing agent and in the presence of an equimolar amount of glucose leads to an increase in the yield of alcohol to 73% and 76%, respectively, however, the optical purity of the alcohol decreases to 62% ee and 80% ee, which suggests the stereoinversion.

Asymmetric Bioreduction of 4-Chloroacetophenone Catalyzed by Daucus carota Cells in Water and Organic Solvents

Asymmetric Bioreduction of 4-Chloroacetophenone Catalyzed by Daucus carota Cells in Water and Organic Solvents

Salt Stress-Induced Anthocyanin Biosynthesis Genes and MATE Transporter Involved in Anthocyanin Accumulation in Daucus carota Cell Culture.

Anthocyanins biosynthesis is a well-studied biosynthesis pathway in Daucus carota. However, the scale-up production at the bioreactor level and transporter involved in accumulation is poorly understood. To increase anthocyanin content and elucidate the molecular mechanism involved in accumulation, we examined D. carota cell culture in flask and bioreactor for 18 days under salt stress (20.0 mM NH4NO3/37.6 mM KNO3) at 3 day intervals. The expression of anthocyanin biosynthesis and putative MATE (multidrug and toxic compound extrusion) transporter expression was analyzed by qRT-PCR. It was observed that there was a significant enhancement of anthocyanin in the bioreactor compared to the control culture. A correlation was observed between the expression of MATE and the anthocyanin biosynthesis genes (CHS, C4H, LDOX, and UFGT) on the 9th day in a bioreactor, where maximum anthocyanin accumulation and expression was detected. We hypothesize the involvement of MATE in transporting anthocyanin to tonoplast in D. carota culture under salt stress.

Enantioselective bioreduction of heptan-2-one and octan-2-one catalyzed by Daucus carota cells

Synthesis of enantiomerically pure (2S)-(+)-heptan-2-ol and (2S)-(+)-octan-2-ol by bioreduction of heptan-2-one and octan-2-one using Daucus carota cells has been studied. Optimization of bioreduction conditions has been carried out in the presence of various exogenous reducing agents (ethanol, isopropanol, glucose). Conditions allowing to obtain (2S)-(+)-heptan-2-ol with 91% yield and optical purity 98% ee and (2S)-(+)-octan-2-ol in 95% yield and > 99% ee have been found.

Relative reactivity of substituted acetophenones in enantioselective biocatalytic reduction catalyzed by plant cells of Daucus carota and Petroselinum crispum

We have examined enantioselective bioreduction of acetophenone and its substituted derivatives into corresponding S-alcohols catalyzed by Daucus carota and Petroselinum crispum plant cells in water and isooctane. We found that the nature of the substituent has a profound effect on the relative reactivity of substituted acetophenones and enantioselectivity of biocatalytic reduction. Electron-withdrawing substituents –Br and –NO2 enhance the initial rate of reaction and yields of products, while electron-donating substituent –OCH3 decreases them. The reduction rates and yields of products in water were noticeably higher in comparison with similar reductions conducted in isooctane. Correlations between the initial reaction rate and the substituent constant (σ+) in the aromatic ring characterizing its nature were established. Comparison of ρ constants of bioreduction catalyzed by D. carota and P. crispum shows that the sensitivity of the reduction to the nature of the substituents is more significant in the case of D. carota biocatalyst. Comparison of ρ constants for D. сarota and P. crispum in water and isooctane indicates that the sensitivity of bioreduction to the nature of the substituent tends to increase from water to isooctane.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168h of treatment (497.0 ± 23.5µgg dry weight-1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota

In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL−1 and 477.46μgL−1, respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL−1), lutein (25949.54μgL−1) and α-tocopherol (8063.82μgL−1) chlorophyll a (1625.13μgL−1) and b (9.958 (9958.33μgL−1) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway.Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

Long and short term effects of plasma treatment on meristematic plant cells

In this paper, we will present results of plasma treatments of meristematic cells of Daucus carota. Plasma needle was used as an atmospheric pressure/gas composition source of non-equilibrium plasma in all treatments. Activity of antioxidant enzymes superoxide dismutase and catalase was measured immediately after plasma treatment and after two weeks following the treatment. Superoxide dismutase activity was increased in samples immediately after the plasma treatment. On the other hand, catalase activity was much higher in treated samples when measured two weeks after plasma treatment. These results show that there is a direct proof of the triggering of signal transduction in the cells by two reactive oxygen species H2O2 and O2−, causing enzyme activity and short and long term effects even during the growth of calli, where the information is passed to newborn cells over the period of two weeks.

Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain

Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.

Use of β-cyclodextrins to enhance phytosterol production in cell suspension cultures of carrot (Daucus carota L.)

Phytosterols are isoprenoid-derived compounds that play essential roles in plant growth and development as they are integral components of the plant cell membranes, and responsible for their permeability and fluidity. In this study, the effect of different types of beta-cyclodextrins (β-CD) on phytosterol production was evaluated using Daucus carota cell suspension cultures. A detailed analysis provides the optimal type and concentration of β-CD, elicitation time, and the optimal cell age and density. The highest levels of phytosterols produced by cells and secreted to the culture medium were obtained when 10 day-old D. carota cell suspension cultures, with an initial cell density of 200 g of fresh weight (FW) l−1, were incubated in the presence of 50 mM of methylated-β-CD (M-β-CD) for 144 h. In addition, M-β-CD significantly promoted the accumulation of phytosterol in the extracellular medium of D. carota cell suspension cultures, which was not observed in control cell suspension cultures. Moreover, these high phytosterol levels did not improve when methyl jasmonate (MJ) was added to the cell suspension cultures alone or combined with 50 mM M-β-CD, although MJ stimulated the formation of defense-related compounds. Therefore, the use of carrot cell suspension cultures seems a promising biotechnological alternative since the addition of β-CD to the culture medium not only induced the biosynthesis of phytosterols but also promoted their secretion into the culture medium, where they were accumulated and could be isolated easily.

Biological effects of water-soluble soil phenol and soil humic extracts on plant systems

The aim of this research was to extract, with a sequential method, HS free from water-soluble phenols (HS-WP) and water-soluble phenols free from humic substances (WP-HS), and to evaluate and to compare the biological effects of these two extracts to humic substances (HS) and water-soluble phenols (WP) traditionally extracted. In each extract we determined the concentration of low molecular weight organic acids, soluble carbohydrates, fatty acids, phenolic acids and total proteins. We tested the biological activity of each soil extract and of the single identified compounds on different plant organs (Pinus laricio callus, Daucus carota cells, and Pinus laricio, Pinus halepensis, Lens culinaris and Cichorium intybus seeds). The results showed that eliminating from HS the WP fraction, and from WP the HS fraction we obtained extracts chemically different from HS as such, and WP as such. HS and HS-WP increased callus and cell growth and also root elongation of the different species used; in contrast, WP and WP-HS had negative effects inhibiting callus and cell growth and seed germination percentage of coniferous, herbaceous and leguminous species. The negative effects can be ascribed to the presence of phenolic acids in the extracts while the positive biological activity can be attributed to the presence of tartaric acid, and fatty acids. In conclusion, this study helps to discriminate the effects of humic substances against phenolic compounds extracted from soils explaining the different and in some way contradictory biological behaviour of these two main fractions of SOM.

Enzymatic preparation of 1-O-hydroxycinnamoyl-.BETA.-D-glucoses and their application to the study of 1-O-hydroxycinnamoyl-.BETA.-D-glucose-dependent acyltransferase in anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis

Four 1-O-hydroxycinnamoyl-β-D-glucoses (HCA-Glcs), sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-glucoses, were synthesized using a recombinant protein of sinapate glucosyltransferase from Gomphrena globosa coupled with a recycling system of UDP-glucose by sucrose synthase from Arabidopsis thaliana. The substrate preference of HCA-Glc-dependent acyltransferase activity was examined in a protein extract prepared from anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis. The main anthocyanin molecule of the aglycon and the sugar moiety produced and accumulated in both cultured cells were exactly the same; the only difference was found in modification with sinapoyl moiety in D. carota and with feruloyl moiety in G. littoralis. The protein extracts from both D. carota and G. littoralis cultured cells showed higher activity with feruloyl-Glc than with sinapoyl-Glc. The major HCA-Glcs that accumulated in cultured cells of D. carota and G. littoralis were sinapoyl-Glc and feruloyl-Glc, respectively. These results suggested that the specificity of HCA moieties of major anthocyanin molecules in cultured cells of D. carota and G. littoralis might be dominated by produced and accumulated acyl donor molecules in vivo rather than by the substrate specificity of acyltransferase enzymes.

Reduced expression of top1 gene induces programmed cell death and alters ascorbate metabolism in Daucus carota cultured cells

Topoisomerase I (topo I) is a nuclear enzyme which plays a fundamental role in several pathways involving changes in DNA topology. The topo I-mediated reaction is accomplished by the transient covalent binding of the enzyme to DNA (topo I-DNA complex). Stabilization of the topo I-DNA complex, leading to irreversible double-strand breaks, has been reported to occur in animal cells under oxidative stress conditions and during apoptosis. In order to study the existence of a putative link between the topo I-mediated DNA damage and ascorbate (ASC) metabolism, also involved in the responses against oxidative stress and in the apoptotic process in plants, Daucus carota cells showing reduced expression of the top1beta gene encoding the topo Ibeta isoform were produced, using an antisense RNA strategy. Two independent transgenic lines (AT1-beta/22 and beta/36), characterized by a slow growth phenotype, resistance to camptothecin, a specific inhibitor of topo I, but sensitivity to etoposide, an inhibitor of topo II, were investigated in this study. In the absence of external stimuli, AT1-beta/22 and beta/36 cells underwent programmed cell death (PCD) in a precocious phase of the growth curve. ASC metabolism showed remarkable differences in AT1-beta/22 and beta/36 cells, compared with control, and the observed alterations were similar to those occurring in tobacco Yellow Bright-2 cells induced to enter PCD by exogenous stimuli. However, differently from other studied examples of PCD, overproduction of reactive oxygen species was not detected in AT1-beta/22 and beta/36 cells. The relevance of these findings in relation to the signalling pathways leading to PCD is discussed.

Daucus carota Cells Contain Specific DNA Methyltransferase Inhibitors that Interfere with Somatic Embryogenesis

Abstract: Results reported in this paper show that carrot cells contain a thermostable inhibiting activity for cytosine‐5‐DNA methyltransferase that, upon filtration chromatography, can be resolved into three major peaks. Inhibiting activity was found in all plant species tested, though at a concentration lower than in carrot. These inhibiting activities differ in size, sensitivity to various hydrolytic treatments, specificity for DNA METases of eukaryotic and bacterial origin and kinetics of inhibition. Results of chemical analyses indicate that the inhibitors differ from lipidic inhibitors described in Escherichia coli and Streptomyces sp. and, given their sensitivity to proteinase K, appear to have a proteinaceous nature. The addition of these inhibitors (Sephadex G25 peak II and peak III) to actively growing suspension rice cells reduced the rate of in vivo DNA methylation without interfering with DNA synthesis. Peak II also induced a general demethylation effect in carrot cell suspension, even if weaker than that caused by 5‐azacytidine. Interestingly, inhibitors suppressed carrot embryogenesis but did not prevent undifferentiated cell proliferation of suspension cultures.

The reticuloplasmin calreticulun is released into the medium by carrot cell cultures

We report here the presence of a 58-kDa protein in the cells of Daucus carota L. cultivated in vitro. Two lines of carrot cells are used: wild-type line (wt) and mutant line (ts11). We describe here also presence of this protein in the media of cultured cells. Strong reaction of this intracellular and extracellular protein with an anti-calreticulin antiserum indicates that it is a major high capacity, low affinity Ca2+-binding reticuloplasmin–calreticulin. No differences in biochemical characterization is found between calreticulin purified from the wild-type line and the mutant line. Moreover molecular mass, type of glycosylation and the ability of extracellular protein to bind calcium is found to be indistinguishable from those of the purified intracellular calreticulin. Calreticulin release is attributed to some stress imposed on cultured cells by growth conditions. It is shown that this process can be also induced in CR-non-releasing systems such as carrot somatic embryos by applying a high-cell-density stress.

Characteristics, hydrolysis of cell wall polymers, and response to calcium deficiency of a cell wall-associated β-galactosidase from carrot cells

Summary The activity of β-galactosidase (EC 3.2.1.23) in the protein fraction solubilized with 3 mol/L LiCl from cell walls of suspension-cultured carrot ( Daucus carota ) cells has been found to be higher than those of the other glycosyl-hydrolytic enzymes. The cell wall-associated β-galactosidase/exo-galactanase was purified to electrophoretic homogeneity. Analysis by denaturing PAGE revealed a single polypeptide chain with a molecular mass of 50 kDa, similar to 52 kDa estimated for the native protein by size-exclusion chromatography. The enzyme contained carbohydrate and protein in a ratio of 1 : 10 (w/w), and was rich in Gly and Phe, followed by Ser, Ala and Thr. The isoelectric point was pH 6.5, and pH and temperature optima 4.4 and 45-50 °C, respectively. The β-galactosidase activity was inhibited by Mg 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate and D-galactono-1,4-lactone. The K m and V max values for p -nitrophenyl-β-D-galactopyranoside were 0.77 mmol/L and 0.32 mmol (mg protein) −1 h −1 , respectively. The enzyme hydrolyzed citrus galactan in an exo-fashion and was capable of hydrolysing an acidic pectic polymer containing galactosyl and arabinosyl residues from carrot cell walls and, therefore, is an exo-galactanase. However, even after an exhaustive reaction, the enzyme had no effect on a galactose-rich hemicellulosic polymer from carrot cell walls. Under calcium deficiency the activities of a cell wall-associated α-galactosidase (EC 3.2.1.22) and β-galactosidase increased until 15 days of culture and then decreased gradually, whereas β-glucosidase (EC 3.2.1.21) activity increased markedly with time despite poor cell growth.

Ensuring oxygenation of carrot cell cultures in a Couette viscometer during investigation of shear effects

Mathematical simulation and experimental measurement of dissolved O2 were performed for extended (up to 8 h) shear testing of Daucus carota (carrot) cell cultures in a conventional Couette viscometer (0.625 mm annulus). The results suggest O2 depletion below critical levels for cell growth may occur. A novel design modification incorporating an O2-permeable silicone-layer spun cast on a porous ceramic bowl was devised. It significantly improved oxygenation of the cell cultures, keeping dissolved O2 near saturation.

Activity of Some Aminopeptidases in Immobilized Cells of Daucus carota

Carrot cell suspension culture prior to immobilization by glutaraldehyde was permeabilised by Tween 80. The values of pH optimum for L-alanine aminopeptidase, L-proline iminopeptidase, and L-arginine aminopeptidase were 8.2, 7.4, and 7.9, respectively. The immobilized cells showed significantly lower aminopeptidase activity when compared to untreated cells. Alginate hydrogel was successfully used for immobilization of carrot cells retaining the activity of some aminopeptidases.

Uptake and phosphorylation of glucose and fructose in Daucus carota cell suspensions are differently regulated

Cell suspensions of Daucus carota L. were grown in batch culture on 50 mM sucrose, 100 mM glucose or 100 mM fructose. Sucrose was rapidly converted extra-cellularly into equimolar amounts of glucose and fructose, and glucose was then taken up preferentially. This impaired uptake of fructose could partially be explained by the eight-fold lower affinity of the hexose carrier in the plasmamembrane for fructose compared to glucose. However, cells grown on fructose as the sole carbon source showed a shorter lag phase and showed more biomass production compared to glucose-grown cells, indicating that conversion of glucose and fructose were also differently regulated. Ninety-five % of the glucose phosphorylating activity was membrane-associated and most probably confined to mitochondria; therefore, it might be present in a respiratory ‘compartment’ making glucose a better substrate for respiration than fructose. The soluble fraction contained the majority of the fructokinase activity. This activity was hypothesized to be more or less randomly distributed through the cytosol; in this soluble ‘compartment’ a pool of fructose-6-phosphate is formed. Concomitantly, via glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P), it is converted into UDPG-glucose, resulting in structural cell components. The observed transient obstruction of the conversion of G-1-P into UDP-glucose in fructose-grown cells, leading to G-1-P accumulation, might be a result of both an altered equilibrium maintained by phosphoglucomutase, interconverting G-6-P and G-1-P and low levels of nucleotide triphosphates. Low nucleotide triphosphate production, connected with a low initial respiration rate, might be caused by the ten-fold lower affinity of the membrane-associated phosphorylating enzymes for fructose compared to glucose. Our results were taken to indicate that two separate pools of glycolytic intermediates exist in D. carota cells: one distributed throughout the cytosol and one surrounding the mitochondria.

Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

Two keratin-like proteins of 64 and 55 ku were purified from suspension cells ofDaucus carota L., and their partial amino acid sequences were determined. The homological analysis showed that the sequence from the 64 ku protein was highly homological to beta-glucosidase, and that from the 55 ku protein had no significant homologue in GenBank. Using conservative sequence of animal IF proteins as primer, we cloned a cDNA fragment fromDaucus carota L. Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves. Homological analysis revealed that it had moderate homology to a variety of alpha-helical proteins. Our results might shed more light on molecular characterization of IF existence in higher plant.