Protoplast Cultures from Cell Suspensions of Daucus carota

Protoplasts, 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.     

Rare trisubstituted sesquiterpenes daucanes from the wild Daucus carota

Phytochemical and biological investigation of the roots of the wild Daucus carota ssp. carota afforded three new and four known compounds, including four sesquiterpenes daucane esters (1-3 [new], and 4), one polyacetylene (5), one sesquiterpene coumarin (6), and sitosterol glucoside. The structures of the new compounds were determined by comprehensive NMR studies, including DEPT, COSY, NOESY, HMQC and HMBC analyses. Based on an agar diffusion assay, 1, 2 and 4-6 were screened and found to contain a range of low antibacterial activities against four gram positive (Staphylococcus aureus, Streptomyces scabies, Bacillus subtilus, Bacillus cereus) and two gram negative species (Pseudomonas aeruginosa, Escherichia coli) as well as antifungal against Fusarium oxysporum and Aspergillus niger using cup agar diffusion assay.     

A permanent ion binding site located between two gates of the Shaker K+ channel.

K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the selectivity filter.     

Hypotensive action of coumarin glycosides from Daucus carota.

Daucus carota (carrot) has been used in traditional medicine to treat hypertension. Activity-directed fractionation of aerial parts of D. carota resulted in the isolation of two cumarin glycosides coded as DC-2 and DC-3. Intravenous administration of these compounds caused a dose-dependent (1-10 mg/kg) fall in arterial blood pressure in normotensive anaesthetised rats. In the in vitro studies, both compounds caused a dose-dependent (10-200 microg/ml) inhibitory effect on spontaneously beating guinea pig atria as well as on the K+ -induced contractions of rabbit aorta at similar concentrations. These results indicate that DC-2 and DC-3 may be acting through blockade of calcium channels and this effect may be responsible for the blood pressure lowering effect of the compounds observed in the in vivo studies.     

Anthocyanins from cell suspension cultures of Daucus carota.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2'-O-beta-D-xylopyranosyl-6'-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.     

胡萝卜DcPAB基因的分离及其结构与功能分析

运用改进的减法杂交技术分离到胡萝卜Poly(A)结合蛋白基因DcPAB .其cDNA编码区长度为 1 977bp ,编码 6 5 8个氨基酸和 1个终止密码子 .基因组转录序列区长度为 4 6 1 6bp ,包含 9个外显子和 8个内含子 .DcPAB在胡萝卜基因组中为单拷贝基因 .该基因在胡萝卜体细胞胚中特异性表达 ,且其表达活性在调控 解调控前后有明显差异 .体外结合实验表明 ,在大肠杆菌中表达并纯化的DcPAB蛋白具有与oligo(A) 2 0 特异性结合的性能 .酵母突变体互补实验进一步证明 ,该基因可以互补PAB基因缺失的酵母突变体的功能缺陷     

Two new eudesmane sesquiterpenoids from Daucus carota L.

Two new eudesmane sesquiterpenoids were isolated from the fruits of Daucus carota L. The structures were established based on the interpretation of high-resolution MS and 1D- and 2D-NMR data, the absolute configuration of compound 1 was determined by single-crystal X-ray diffraction. The new compounds were elucidated as (1βH, 3βH, 5βH, 7αH)-4α, 10α-dimethyl-7β-isopropyl-1α, 3α, 4β, 11-tetrahydroxy decahydronaphthalene (1) and (1βH, 3βH, 5βH, 7αH)-4α, 10α-dimethyl-7β-isopropyl-1α, 3α, 4β-trihydroxy decahydronaphthalene-11-O-β-d-glucopyranoside (2). The compound 1 showed significant antioxidant activity.     

The greening of chromoplasts in Daucus carota L.

Summary Evidence was obtained for the transformation of chromoplasts to chloroplasts in the cortex parenchyma of carrot during exposure to light. Typical chromoplasts containing carotene crystals but no lamellar system were observed at the onset of illumination. The ensuing synthesis of chlorophyll and a lamellar system was accompanied by disappearance of the carotene crystals. Only chloroplasts were present after 48 hr in the light. The different stages of plastid development were observed using the electron microscope.     

Chalcone synthase from cell suspension cultures of Daucus carota L

Chalcone synthase (CHS) has been partially purified about 35-fold. Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps. In order to determine CHS activity, two procedures [according to Schr?der et al. (1979) Plant Sci. Lett. 14, 281-286] were applied. The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC. The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay. Both effects were synergistic, but BSA did not promote "side products" as 2-mercaptoethanol did. BSA (10 mg ml-1) and 2-mercaptoethanol (1.4 mM) were components of the standard assay. Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8). The apparent Km values were 0.6 microM for 4-coumaroyl-CoA (pH 7.9), 7.7 microM for caffeoyl-CoA (pH 6.8), and 3.0 microM for malonyl-CoA (pH 7.9). Substrate inhibition was observed with 4-coumaroyl-CoA (greater than 10 microM) and malonyl-CoA (greater than 50 microM). The inhibitory activity of various flavonoids and related compounds (100 microM) was investigated. Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50%. In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40%. It was shown that the inhibition with naringenin was fully uncompetitive. These in vitro data support the view that the true substrate of CHS in D. carota is 4-coumaroyl-CoA.     

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.     

Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg²⁺ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC₅₀ 32 mmol m^–3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

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.     

Cavity spot of carrot (Daucus carota)

Cavity spot disease of carrot (Daucus carota) has been one of the intractable problems for both growers and scientists. Carrots are rejected at grading with one or two visible lesions, and when disease incidence passes a relatively low threshold it becomes uneconomic to harvest crops. For the scientist, there has been considerable pressure to produce both information on the cause of the disease and a cure. Many putative causes have been advanced over the years, but these were almost always contradicted by subsequent work. The first solid indication of involvement of a pathogen was when three different fungicides with activity against Oomycete fungi all reduced disease. Very quickly the causal agents Pythium violae and Pythium sulcatum were isolated from cavity spot lesions and Koch's postulates satisfied. The species are not typical of the more common pythia, having slow growth at normal temperatures, which means that in the context of isolation work, plates may be overgrown by other species before they are seen. Metalaxyl fungicide was identified as the most effective in controlling cavity spot caused by P. violae, but P. sulcatum is naturally tolerant of the fungicide. Recently, metalaxyl has been shown to be subject to enhanced microbial degradation. This phenomenon has been associated with failure to control cavity spot. No other fungicide has been shown to be consistently effective in the field, and none has been registered for disease control. For the future, this means that control of cavity spot can not be based solely on fungicidal control. Other, complementary strategies are necessary for reducing disease. Calcium carbonate is known to have significant effects on cavity spot, probably by inducing a soil microflora inhibitory to filamentous fungi. Management of agronomic aspects such as irrigation, soil cultivation and the length of time for which crops are grown may all be used, while carrot cultivars with some field resistance may be beneficial. However, one of the most significant factors is disease avoidance by not selecting fields with high inoculum levels. One serology‐based risk assessment test has been produced and commercialised, and molecular probes which could be the basis of more sensitive tests are available for both pathogens. The potential for disease reduction via a management strategy combining several key components is discussed.     

Ontogeny of Daucus carota Infected with Meloidogyne hapla

The ontogeny of carrots (Daucus carota cv. ''Spartan Premium'') grown under greenhouse conditions in pots of organic soil infected with Meloidogyne hapla was influenced detrimentally as early as 4 days after seeding, as determined through analysis of plant surface area, dry weight, fresh weight, net assimilation rate, relative growth rate, and leaf-area ratio. Only 58% of the diseased carrots were suitable for fresh market, compared with 97% of those grown in nematode-free soil. Growth and development of the shoot system (height, surface area, dry weight, and fresh weight) were retarded by M. hapla as early as 12 days after seeding. During the first 12 days after seeding, root dry weight was greater for diseased plants than for controls. Root growth and development (surface area, dry weight, and fresh weight) associated with this nematode, however, were retarded as early as 16 days after seeding. M. hapla caused a delay in the occurrence of 2nd-, 4th-, and 5th-order roots, and an increase in the occurrence of 6th-order roots in infected plants. Parasitized plants had 44% fewer roots (primary through 6th-order) and 50% less total root length.