Isolation and Characterization of a Carrot Cell Line Resistant to Methotrexate

A carrot cell line (WCA1) resistant to Mtx has been isolatedand partially characterized. The IC₅₀-Mtx is about thirty timeshigher in the resistant than in the parental line and the rateof uptake of Mtx is decreased 15 times. In addition the specificactivity of DHFR is about three times higher in the resistantline as compared to the parent Key words: Methotrexate resistance, Dihydrofolate reductase, Methotrexate uptake, Plant cell suspension culture, Daucus carota     

Salinity, oxidative stress and antioxidant responses in shoot cultures of rice

When shoot cultures derived from salt-sensitive Oryza sativavar. Taipei 309 were grown at 25C in medium containing 0.35M NaCl, responses to possible oxidative stress in the earlystages of exposure were observed. Overall levels of Mn-superoxidedismutase activity, Cu, Zn-superoxide dismutase activity andH₂O₂ were significantly elevated. After 1 d there was a notabledecline in tissue concentrations of GSH and a correspondingincrease in GSSG. However, after a further day, concentrationsof GSH and GSSG returned to concentrations normally encounteredin control cultures. Activities of ascorbate peroxidase andcatalase were similar whether the shoots were grown in the presenceor absence of NaCl. In contrast, there was an early increasein glutathione reductase activity in NaCl-exposed cultures,and no indication of extensive increases in lipid peroxidation.Thus although some indications of oxidative stress accompanyexposure of this salt-sensitive rice variety to salinity, mechanismsappear to exist within its shoot tissue to permit the toleranceof such oxidative stress. Key words: Salinity stress, hydrogen peroxide, glutathione, antioxidant enzymes, Oryza sativa     

Intercellular Deformation in Compressed Organs

Intercellular deformations, caused by increasing levels of compressionapplied by a pressure chamber to an organ covered with a plasticsealant and evaluated according to the internal atmosphere removalrate, were observed in carrots (Daucus carota L. sativa), potatoes(Solanum tuberosum L.) and sweet-potatoes (Ipomea batatas L.Lam). The maximum internal gas volume removed in these kineticassays was close to the intercellular air volume (V_g) measuredby the pycnometric method. Presumably a compression larger thanthe average organ turgor was required to remove all V_g and abovethis point the cells should become completely flattened againsteach other. The intercellular deformation caused by a compressingload, observed by constant pressure volumetry, induced a reductionin the endogenous O₂ concentration at the stressed area, accordingto polarographic measurements. Cellular deformations and eventualV_g flooding caused by water movement from the symplasm to theapoplasm of externally compressed organs were distinct fromthe usual pressure chamber assays, where all cells are exposedto homogeneous gas pressurization, without the development offorces to cause large cellular deformation and intercellularflooding. These gas transport restrictions were suggested aspotential causes for post harvest deterioration in fragile commoditiessubjected to compression.Copyright 1995, 1999 Academic Press Carrot, compression, Daucus carota, gas volumetry, Ipomea batatas, oxygen, porosity, potato, pressure chamber, Solanum tuberosum, stress, sweet-potato, turgor, suction     

Glucose and Pyruvate Metabolism in Daucus carota Cells: The Effect of the Ammonium Ion

In Daucus carota cells cultivated in vitro, the ammonium ionstimulates the incorporation of radioactivity from labelledglucose and labelled pyruvate into CO₂ and into the residueinsoluble in 60 per cent (v/v) ethanol. There is a higher ¹⁴CO₂production from [6-¹⁴C₂] glucose than from [6-¹⁴C] glucose.These results suggest a possible stimulation of glycolysis bythe ammonium ion.     

The Effect of Inorganic Nutrients on the Hormonal Requirements of Normal, Habituated, and Crown-gall Tissue 4

The addition of Braun and Wood's inorganic supplements (845mg l^–1 KCl, 1800 mgl^–1 NaNO₃, 300 mg l^–1 NaH₂PO₄.2H₂O,790 mg l^–1 (NH₄)₂SO₄) to White's medium caused markedincreases in the growth of normal tissues of Helianthus annuus,Nicotiana rustica, Daucus carota, and Vinca rosea and crown-galltumour tissues of H. annuus. However, no evidence was obtainedwhich suggested that the presence of these extra salts markedlyinfluenced the essential requirements of normal callus for auxinsand kinetin. In contrast their presence significantly influencedthe hormonal requirements of certain habituated cultures ofH. annuus and V. rosea. These habituated cultures had specificauxin requirements on White's medium while either an auxin orkinetin was sufficient on high-salts medium. These results arediscussed in relation to previous reports which suggested thatthe biosyntheses of auxins and other growth factors in normaland crown-gall cultures are specifically activated by certaininorganic ions.     

Characteristics of Aluminum-Phosphate-Adapted Carrot Cells: Uptake and Utilization of the Phosphate

To elucidate the mechanism responsible for the superior growthof a selected line of carrot cells (Daucus carota L. cv MS Yonsun)in medium that contained AIPO₄, kinetic studies of the uptakeof phosphate and the efficiency of utilization of phosphatewere performed with the selected cells and the wild-type cells.When the two cell lines were grown in a medium with adequatesoluble phosphate (2 mM), there was no difference between theirgrowth rates. Rates of increase in fresh weight as a functionof increasing concentration of phosphate in the medium werealso identical between the cell lines, indicating that the efficiencyof utilization of phosphate by the selected cell line was similarto that by the wild-type cells. However, rate of uptake of phosphateby the selected cells under phosphate limited conditions (20µMNaH₂PO₄ at pH 5.6) was about 5-fold higher than that by thewild-type cells. Apparent K_m values for the uptake of phosphatewere calculated to be 13.6 and 9.1 µM for the selectedand the wild-type cells, respectively. The V_max valuewas estimatedto be 88.8 nmol per g fresh weight per min for the selectedcells and 28.2 for thewild-type cells. Thus, the selected cellshas an enhanced system for uptake of phosphate wherebytherewas an increase in the rate of the uptake without any dramaticchange in the affinity for phosphateions. (Received September 21, 1991; Accepted December 25, 1991)     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

Effect of Folate Analogues on the Activity of Dihydrofolate Reductase and Callus Growth of Sunflower

The inhibitory effect of antifolate analogues in vitro and invivo on dihydrofolate reductase and on growth of sunflower callus,was tested. Aminopterin and trimethoprime proved to be the mosteffective inhibitors. The ID₅₀ values for these compounds werethe same as those previously shown for enzymes from rice andcarrot cells. Protein, DNA and RNA synthesis of sunflower callus was inhibitedby the analogues, the inhibition depending on the concentrationused. These results suggest that growth inhibition of sunflowercallus by these analogues is due to inhibition of dihydrofolatereductase. Key words: Antifolate, callus, dihydrofolate reductase, growth, sunflower     

The Metabolic Fate of (4-Chloro-2-Methylphenoxy)Acetic Acid in Higher Plants: I. GENERAL RESULTS

The metabolic fate of the auxin herbicide (4-chloro-2-methylphenoxy)aceticacid (MCPA) has been determined in a number of species usinga vacuum infiltration technique. In all cases MCPA became hydroxylatedto form (4-chloro-2-hydroxymethylphenoxy)acetic acid, whichaccumulated largely as a glycosidic conjugate. The nature ofthe oxidized metabolite from oat (Avena sativa L.) was verifiedby GC/MS. In all cases at least one diethyl ether-soluble conjugateof MCPA was formed; these are suggested to be amino acid conjugates.Several minor aglycones were also formed. Important speciesvariations in both the rate and quantitative nature of metabolismwere observed. Pretreatment of potato (Solarium tuberosum L.)tuber slices with unlabelled MCPA and other auxins increasedthe capacity for hydroxylation, but particularly induced theformation of an MCPA-glycoside. This was never a major metaboliteunder normal circumstances. The rate of hydroxylation was alsoenhanced by ageing in MnCl₂. Although the ether-soluble conjugatesof MCPA were stable metabolites, exogenously applied conjugateisolated from carrot (Daucus carota L.) was readily cleavedin four species. Free MCPA and the products normally derivedfrom it were identified. MCPA Metabolism Hydroxylation Conjugation     

Transgenic Rice: Characterization of Protoplastderived Plants and their Seed Progeny

Thirty eight green and 2 albino plants were regenerated from400 kanamycin-resistant colonies derived from protoplasts isolatedfrom cell suspensions of Oryza sativa variety Taipei 309 andelectroporated with pCaMVNEO carrying the neomycin phosphotransferaseII (nptII) gene. Twenty of the green transgenic R_o plants weretransferred to the glasshouse, where 3 flowered after 7 months.Of 15 plants analysed by DNA hybridization, all carried thenptll gene, but only 2 of 11 plants assayed for NPTII activityexpressed the nptll gene. One transgenic R_o plant produced 59seeds following self-pollination. The seeds, when germinatedon medium containing kanamycin sulphate, gave 16 green transgenicR, plants. Five transgenic R₁ plants flowered and set seed,7 flowered but failed to produce seeds, while 4 did not producepanicles. Transgenic R_o and R₁ plants were shorter, requiredlonger to flower, and had reduced pollen viability comparedto non-transformed R₁ protoplast-derived plants. The nptII genewas present in all 16 transgenic R₁ plants, but NPTII activitywas detected in only 8 of these plants. Key words: Oryza sativa variety Taipei 309, rice, protoplasts, direct DNA uptake, kanamycin-resistant tissues, transgenic plants, DNA hybridization, neomycin phosphotransferase II (NPTII), gene expression and inheritance     

Thymidylate Synthase in Plant Cells: Kinetic and Molecular Properties of the Enzyme from Daucus carota L. Cell Cultures

Thymidylate synthase activity was assayed in cell homogenatesfrom wild or domestic carrot (Daucus carota L.) suspension culturesusing the tritium-release method. The specific activity measuredwas lower than that previously reported for bacterial extracts,and its level did not change a great deal during a 14 day growthcycle. The enzyme was partially purified (about 9 fold) by ammoniumsulfate fractionation and ion-exchange chromatography. Kineticparameters such as pH optimum, K_m values for substrate and cofactor,and degree of inhibition by 5-fluoro-2'-deoxyuridine monophosphatewere characterized. The apparent mol wt, evaluated by gel-filtration chromatography,was 185 kDa, which was much higher than that reported for enzymesof bacterial and vertebrate origins and somewhat higher thanthat reported for the thymidylate synthase-dihydrofolate reductasebifunctional polypeptide of some protozoa. (Received April 20, 1987; Accepted February 15, 1988)     

Energetics of Plant Growth Under Anoxia: Metabolic Adaptations of Oryza sativa and Echinochloa phyllopogon

Unlike most plant species, Oryza sativa L. cv. S-201 and Echinochloaphyllopogon (Stev.) Koss germinate and grow under anaerobicconditions. In both species, the radicle or shoot emerged byday 3 when the seeds were germinated in air or N₂. Under eithercondition, shoot and/or root dry weight (d. wt) increased linearlyfrom day 3 to day 7, with a corresponding decrease in seed d.wt. In anaerobically grown O. sativa, d. wt accumulation wasreduced to 7% of that in air whereas d. wt lost from the seedwas reduced to only 37%. No root growth occurred during anaerobicgermination and shoot d. wt accumulation accounted for 10% ofthe d. wt lost from the seed. In E. phyllopogon, d. wt accumulationduring anoxia was 25% of that in air, but loss of d. wt fromthe seed was 44% of the aerobic rate. In air, 48% of the d.wt lost from the seed was converted to shoot or root d. wt.Like O. sativa, E. phyllopogon does not produce a root underN₂, but shoot growth accounted for 27% of the d. wt lost fromthe seed. Thus, either in air or N₂, E. phyllopogon was moreefficient at converting seed reserves to shoot/root structuraldry matter than O. sativa . Based on changes in metabolite pools,O. sativa appeared to shift exclusively to fermentation duringanaerobic growth. In E. phyllopogon, however, fermentation alonecannot satisfy the energy requirement for growth without O₂.Rather, fermentation, coupled with limited tricarboxylic acid(TCA) cycle operation could supply sufficient ATP for growthunder anaerobic conditions. An active oxidative pentose phosphatepathway and lipid synthesis were discussed as important mechanismsfor converting NADH to NAD, a necessary cofactor for fermentationand TCA cycle activity.Copyright 1994, 1999 Academic Press Anaerobiosis, Echinochloa phyllopogon, energetics model, fermentation, mitochondrial activity, Oryza sativa, rice, tricarboxylic acid cycle, watergrass     

Photosynthesis and Transpiration in Rice as Influenced by Soil Moisture and Air Humidity

The paper describes the effect of soil moisture content andair humidity on CO₂ exchange (P_N), CO₂ diffusion resistance(Cr) and transpiration (E) in four varieties of japonica rice(Oryza sativa L.). A decrease in soil moisture content reducedthe rate of photosynthesis to a varying degree in the varieties.Reduction in photosynthesis was attributed to increase in Cr.The effect of low soil moisture on photosynthesis and CO₂ diffusionwas further intensified by decrease in air humidity. By maintaininga high humidity in the air around the leaves however, the effectof soil moisture deficiency was reduced considerably, exceptin Rikuto Norin 21 which was very sensitive to soil-moisturedeficiency alone. Dryness of the air enhanced the transpirationrate, although the increase was relatively less in the plantsfacing a simultaneous water crisis at the root surface. In plantsgrowing under flooded conditions, a decrease in air humiditycaused a slight depression in P_N despite the simultaneous decreasein Cr. Oryza sativa L., rice, photosynthesis, transpiration, diffusion resistance, soil moisture, air humidity     

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     

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin. Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.     

Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.     

Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells

Batch suspension cultures of chicory cells (Cichorium intybusL. var. Witloof) possess a NADH-specific nitrate reductase activitythat peaks on day 3 of a 10 d growth cycle. When both nitrateand ammonium are used as nitrogen sources, chicory cells absorbnitrate irst. Ammonium uptake becomes predominant at day 3,even though NO₃^– was still present in the medium. Althoughabscisic acid impairs growth as well as ¹⁵NO₃^– uptakeand reduction, it promotes nitrate reductase activity as measuredboth in vivo and in vitro. Specific activity is 50% higher inABA-treated cells than in controls. These conflicting data maybe explained either in erms of nitrate reductase levels or bythe availability of reducing power and energy. Since NRA isgenerally controlled by the availability of the reducing power,the energy status of the cell, the adenylate nucleotide pools,were measured simultaneously with the carbohydrate levels withinthe cell and the growth medium. The energy charge was not modifiedduring the growth cycle, regardless of the rowth conditions.Yet ABA modified the intracellular carbohydrate metabolism andinhibited the acidic invertase, the sucrose synthase and thesucrose phosphate synthase activities. Modified assimilationrates of nitrate in chicory cells grown in the presence of ABA,were probably correlated to modified carbohydrate metabolismpathways leading to increased availability of reducing power,energy and C-skeletons. Key words: Abscisic acid, Cichorium intybus L, nitrate reductase, reductase, invertase, sucrose synthase, sucrose phosphate synthase     

Dynamics of Competition in Populations of Carrot (Daucus carota)

Populations of carrot (Daucus carota) were raised over a widerange of densities (79–5763 plants m^-2) to examine thedynamics of competition in terms of yield–density relationshipsand size variability, and to investigate the effects of nutrientsupply on competition. While the relationship between shootyield and density was asymptotic, the relationship between rootand total yield and density tended to be parabolic. For a giventime and density series the relationship between yield per unitarea and density could best be described by the model: y=w_mD_(1+aD)^b wherey is the yield per unit area,D is density,w_m, a andb arefitted parameters. The parametersw_m anda increased over timebut nutrient availability affected onlyw_m. An extension of thebasic yield-density model is proposed to describe the dynamicsof the yield–density relationship over time: y=kD_{[1+cexp(-rt)]{1+}