Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Vegetable Plant Part Relationships. I. Effects of Time and Population Density on the Shoot and Storage Root Weights of Carrot (Daucus carota L.)

Chantenay carrot plants were grown in replicated plots containingdensities of 25, 64, 130 and 245 plants m^–2 and harvestedon 20 weekly occasions giving a total of 360 pairs of d. wtsand 13908 pairs of f. wts of shoots (s) and roots (r). The relationshipbetween the logarithms of s and r was curved for plants sampledon successive occasions from the same density but it was linearfor plants sampled on a single occasion, both within and betweendensities. A good overall relationship was In s = a+0.805 In r, where the intercept (a) ranged in approximately equal stepsfrom +1.2 to –1.1 at the first and last harvests respectively.Except for some systematic deviation from this equation at earlyharvests, the above relationship fitted the data well irrespectiveof the wide range of density and whole plant weight. Daucus carota L. carrot, root/shoot weight, population density, growth analysis     

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     

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Pigeonpea is a tropical grain-legume, which is highly dehydrationtolerant. The effect of drought stress on the carbohydrate metabolismin mature pigeonpea leaves was investigated by withholding waterfrom plants grown in very large pots (50 kg of soil). The moststriking feature of drought-stressed plants was the pronouncedaccumulation of D-pinitol (1D-3-methyl-chiro-inositol), whichincreased from 14 to 85 mg g^–1 dry weight during a 27d stress period. Concomitantly, the levels of starch, sucroseand the pinitol precursors myo-inositol and ononitol all decreasedrapidly to zero or near-zero in response to drought. The levelsof glucose and fructose increased moderately. Drought stressinduced a pronounced increase of the activities of enzymes hydrolysingsoluble starch (amylases) and sucrose (invertase and sucrosesynthase). The two anabolic enzymes sucrose phosphate synthase(sucrose synthetic pathway) and myo-inositol methyl transferase(pinitol synthetic pathway) also showed an increase of activityduring stress. These results indicate that pinitol accumulatedin pigeonpea leaves, because the carbon flux was diverted fromstarch and sucrose into polyols. Key words: Drought, polyols, pinitol, sucrose, starch, pigeonpea     

Vegetable Plant Part Relationships. III. Modification of Carrot (Daucus carota L.) Root and Shoot Weights by Gibberellic Acid and Daminozide

Aqueous foliar sprays of N-dimethylaminosuccinamic acid (daminozide)at 2000 p.p.m. and gibberellic acid (GA) at 100 p.p.m. wereapplied 45, 59, 82 and 100 days after sowing to Chantenay carrotswith population densities of 244, 495 and 883 plants m^–2.The plants were harvested on ten approximately weekly occasions;fresh weights were determined and d. wt estimates were obtainedfor the separated shoots (s) and roots (r). Allometric linearregressions of the logarithm of s on that of r at each harvestseparately, clearly showed that GA always increased shoot: rootratio and reduced root yield (by approximately 35 per cent)but could sometimes also increase whole-plant weight. Daminozideincreased root yield (by approximately 7 per cent from 80 tonnesha^–1) and tended to have effects opposite to those ofGA. Daucus carota L., carrot, root weight, shoot weight, N-dimethylaminosuccinamic acid (daminozide), gibberellic acid     

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+}     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Isolation of a Type I Topoisomerase from Carrot Cells

Carbonera, D., Cella, R., Montecucco, A. and Ciarrocchi, G.1988. Isolation of a type I topoisomerase from carrot cells.—J.exp. Bot. 39: 70–78. A DNA topoisomerase activity has been isolated from suspension-culturedcells of Daucus carota, the enzyme has been chromatographedon CM-cellulose, DNA-cellulose and Sephadex G100. Its Mr appearsto be about 100000 by gel filtration. Carrot DNA topoisomeraserelaxes both positively and negatively supercoiled DNA by changingthe linking number of the substrate in steps of one and is notable to unknot the knotted P4 DNA. It does not require ATP orMg²⁺ , has an optimal salt concentration between 40 and 120mol m^–3 KCl and is resistant to nalidixic acid and novobiocine.This carrot enzyme can be designated as a eukaryotic type IDNA topoisomerase. Key words: DNA topoisomerase, Daucus carota     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

Partition of 14C Assimilate between Organs and Fractions of Contrasting Varieties of Carrot during Initiation of the Storage Root

This work examines the differences in partition and activityof ¹⁴C in two varieties of carrot (Daucus carota L.) contrastingin shoot to storage root ratio at maturity. Plants were grownin a controlled environment of 20 ?C and 500 µmol m^–2s^–1. During initiation of the storage root (10–25d from sowing) plants were exposed to ¹⁴CO₂ for 1 h and theradioactivity in ethanol-soluble and -insoluble fractions ofshoots, storage and fibrous roots estimated at various timesup to 48 h after exposure. Between 35% and 40% of radioactivityinitially present in the plants was respired during the first24 h and 25–35% of that remaining after 24 h was foundin the roots, depending on age. The proportion found in thestorage region remained fairly constant between 15 and 25 dand was smaller than at 10 d. In the variety with a larger proportionof storage root at maturity (cv. Super Sprite), there was agreater proportion of label in both ethanol-soluble and -insolublefractions of the storage region soon after storage root initiationhad begun than in the variety with a smaller proportion of storageroot at maturity (cv. Kingston). There was no varietal differencein specific activities of the storage roots, but fibrous rootsof cv. Super Sprite showed a greater specific activity thanin cv. Kingston. Differences in shoot to storage root ratiomay thus be associated with characteristics of the fibrous roots.Partition and specific activities are discussed in relationto the initiation and development of the storage organ. Key words: Daucus carota, carrot, assimilate, partition, ¹⁴C, storage root     

Co-culture with Daucus carota somatic embryos reveals high 2,4-D uptake and release rates of Arabidopsis thaliana cultured cells

By means of co-culture in growth regulator-free medium we analysed whether factors secreted into the medium of Daucus carota (carrot) somatic embryo cultures would be able to overcome the developmental arrest of globular Arabidopsis thaliana somatic embryos. Instead of Arabidopsis embryogenesis being promoted the development of carrot somatic embryos was inhibited at the globular stage in the presence of Arabidopsis suspension culture aggregates with attached globular embryos. Several experiments showed that this was due to the release of previously accumulated 2,4-D by the Arabidopsis cultures. (1) In addition to arresting carrot embryogenesis, co-culture with Arabidopsis cell suspensions also induced callus formation on Arabidopsis root segments. (2) Both effects only occurred with Arabidopsis suspensions grown in the presence of 2,4-D and not with those grown in the presence of NAA, demonstrating that Arabidopsis is not segregating a “general” inhibiting factor. (3) Both effects could be prevented by either binding 2,4-D to active charcoal or by washing it away by changing the medium daily. (4) Uptake of 2,4-D into Arabidopsis cells during culture in 2,4-D containing medium and subsequent release of 2,4-D after transfer to growth regulator-free medium was measured. (5) These low levels of released 2,4-D (0.2– 0.5 μm) could mimic the observed effects. Taken together these data suggest that the high intracellular 2,4-D content of Arabidopsis cultures may interfere with Arabidopsis somatic embryo development beyond the globular stage. Received: 13 November 1997 / Revision received: 2 February 1998 / Accepted: 16 November 1998     

Ethylene and Ethane Production in 2,4-D Treated and Salt Treated Tobacco Tissue Cultures

Gas chromatography was used to measure ethylene (ethene) andethane production by tobacco (Nicotiana tabacum cv. Wisconsinno. 38) callus tissues grown on media containing inorganic saltsaccording to Murashige and Skoog (1962), sucrose, myo-inositol,thiamine-HCl kinetic according to Linsmaier and Skoog (1965),and either 2,4-dichiorophenoxyacetic acid (2,4-D) in the range0–100 mgl^–1 or 2 mgl^–1 indoi-3-ylacetic acidplus NaCl in the range 0–200 Meq l^–1. Ethylene productionrates were high (> 500 nl h^–1 g^1– fresh weight)initially in all treatments. Subsequently, ethylene productiondeclined in rapidly growing cultures but remained high in moderatelyand severely 2,4-D (> 0·5 mgl^–1) stressed andin severely NaCl (150 Meql^–1) stressed cultures. Highinitial rates of ethane production (> 200 nl h^–1 g^–1fresh weight) were obtained under conditions of severe stresscaused by 2,4-D or NaCl but not in control or moderately inhibitedcultures. With further incubation ethane production declinedin the severely stressed cultures. It is concluded that ethyleneproduction can be used as an index of moderate 2,4-D stressand severe NaCl stress by virtue of the high persisting ratesof ethylene production in stressed cultures. Ethane productioncan be used as an early index of severe stress caused by either2,4-D or NaCl in vitro. Nicotiana tabacum L., tobacco, ethylene, ethenen, ethane, 2,4-dichlorophenoxyacetic acid, auxin, stress, callus tissue     

Changes in Levels and Integrity of Ribosomal RNA During Seed Maturation and Germination in Carrot (Daucus carota L.)

Thompson, S., Bryant, J. A. and Brocklehurst, P. A. 1987. Changesin levels and integrity of ribosomal RNA during seed maturationand germination in carrot (Daucus carota L.).—J. exp.Bot. 38: 1343–1350. Amounts and integrity (percentage of complete un-nicked molecules)of ribosomal RNA (rRNA) during germination in carrot seed lotsof differing vigour and viability were examined using aqueousand formamide gel electrophoresis. In unaged seed, amounts ofrRNA per seed in dry seed are not related to maturity, vigouror viability. However, rRNA in immature seed is more vulnerableto damage by ageing than rRNA in mature seed; thus, amountsof rRNA per seed in aged seed are indicators of vigour and viability.On imbibition of aged or unaged seed, more rRNA fragmentationoccurs in immature seed than in mature seed, and the time atwhich the effects of synthesis outweigh the effects of degradationis earlier during germination in mature than in immature seed. Key words: rRNA, seed maturation, seed germination, Daucus carota L.     

Effects of ABA on Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl I. Changes in Incorporation of Glucose and myo-Inositol into Cell-Wall Components

Externally supplied [³H]myo-inositol and [¹⁴C]glucose were incorporatedin cell-wall fractions of segments of etiolated squash hypocotyl.The extent of incorporation of [¹⁴C]glucose into cell-wall fractionswas very much greater than that of [³H]myo-inositol. Radioactivityfrom [¹⁴C]-glucose was effectively incorporated into hemicelluloseB and cellulose fractions and was incorporated uniformly intohexose, pentose and uronic acid residues, but radioactivityfrom [³H]myo-inositol was incorporated predominantly into uronicacid and pentose residues in the pectin and hemicellulose Bfractions. Exogenously applied ABA significantly suppressed the elongationof segments of squash hypocotyl and the incorporation of radioactivityfrom [^l4C]glucose and [³H]myo-inositol into the segments. Furthermore,ABA significantly inhibited the distribution of incorporatedradioactivity from [¹⁴C]glucose into the cellulose fraction,but did not affect distribution into the pectic fraction. Bycontrast, ABA only slightly inhibited the distribution of theincorporated radioactivity from [³H]myo-inositol into the pecticfraction. These results suggest that most of the cell-wall polysaccharidesin segments of squash hypocotyl are synthesized via the UDP-sugarpathway, and that ABA significantly inhibits the synthesis ofcellulose but not the synthesis of pectic polysaccharides whenABA suppresses the elongation of the segments. (Received March 25, 1988; Accepted November 15, 1988)     

Electrophoretic variation in glutamate dehydrogenase and other isozymes in wild carrot cells cultured in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Electrophoretic analysis of isozymal differences was performed with extracts of wild carrot (Daucus carota L.) cells, grown in the presence and absence of 2,4-dichlorophenoxyacetic acid (2,4-D). There were no differences in the patterns of malate dehydrogenase, acid phosphatase, aspartate aminotransferase, and γ-glutamyl transferase. Quantitative differences in peroxidase isozymes were detected, the plus 2,4-D cultures having lower activities. Esterase patterns were similar, but there were differences in individual isozyme activities and an additional form present in the minus 2,4-D cells. the greatest differences were in patterns of glutamate dehydrogenase with the minus 2,4-D cultures containing only the slowly migrating isozymes. The changes in glutamate dehydrogenase, as revealed by isozyme changes, together with the requirement for ammonia in embryogenesis, suggests that this enzyme may be associated with differentiation in wild carrot cells.     

Potential Yield in Carrots (Daucus carota L.): Theory, Test, and an Application

There is little published information on the physiological behaviourof carrots at the crop level. Here we derive and test a simplemodel for the potential yield of carrot crops. The model calculatesgreen leaf area index (L) using a daily time step. Dry matterproduction is related linearly to light interception, calculatedfromL and canopy light extinction coefficient (k). Two stagesof growth are distinguished. In stage 1, leaf expansion on eachplant is unaffected by neighbouring plants. Stage 2 commenceswhen L reaches a critical value and the plants start to interact.Compared to stage 1, stage 2 has slower leaf expansion and ak which varies with plant density. Dry matter partitioning betweenshoots and the storage root depends on L. We calibrated themodel for two processing cultivars, ‘Chantenay Red Core’and ‘Red Hot’, using data from a 1997–98 plantdensity experiment in Hawke's Bay, New Zealand. The model accountedfor 72% of the observed variation in root size and 79% of thevariation in yield. We tested the model against results fromtwo experiments in 1995–96 and 1996–97. In bothexperiments the same two cultivars were sown at three differentsowing times. Overall, the model accounted for 72% of the observedvariation in root size and 66% of the variation in yield, showingthat it is portable to other environments. Finally, we appliedthe model to interpret the effects of sowing date in these twoexperiments. Previous attempts were confounded by variationin plants m^-2with sowing date. The model allowed us to separatethe effects of these factors, and indicated that early sowingsubstantially benefited yield. Copyright 2000 Annals of BotanyCompany Carrot, Daucus carota L., day-degrees, genetic algorithm, growth modelling, plant density, potential yield, thermal time     

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.     

A New Model Relating Crop Yield and Plant Arrangement

A new model is proposed which relates the weight of plants totheir spatial arrangement. The weight of each plant is calculatedas the integral of the function f(r) = L(cr² + 1)^–2 overan area allocated to it, r being distance from the plant, withL and c parameters to be specified. The model is thus concise,general, in that it can be used to describe the effects of anyspatial arrangement on plant weight, and the parameters L andc have a biological interpretation. It is also consistent withthe commonly-used relationship between plant weight (w) anddensity (p), w^–1 = a+bp. We show for carrots (Daucus carota L.) and red beet (Beta vulgarisL.), that the mean weights fitted by the model agree as wellwith the experimentally observed mean plot weight as those fittedby more complex models with more parameters, some of which arenot as general. We show also that the parameter c can be predictedfrom the time from sowing to harvest, with good results whentested on sets of data independent of those to which the modelhad been fitted. The assumptions on which the model is based,its application, and extensions to it are discussed. Crop yield, plant density, plant arrangement, carrot, Daucus carota L., red beet, Beta vulgaris L., soya bean, Glycine max L., mathematical model     

Effects of High and Very Low Magnetic Fields on the Growth of Hairy Roots of Daucus carota and Atropa belladonna

Hairy roots of Daucus carota and Atropa belladonna, which wereinduced by inoculation with Agrobacterium rhizogenes that harboredthe Ri plasmid, were cultured on Murashige and Skoog's solidmedium in magnetic fields of 5 kgauss or 50 µgauss. Thegrowth rate of roots exposed to 5 kgauss was 25% greater thanthat of the control (0.01 kgauss). In the case of A. belladonna,the growth rate of the roots cultured in a field of 50 µgausswas 40 to 56% greater than that of the control (0.5 gauss).In D. carota, after more than 40 h of incubation, growth ratesin both sets of conditions (50 µgauss and control) werethe same. ⁴Present address: Biological Laboratory, Faculty of Education,Yamagata University, Yamagata, 990 Japan. (Received August 24, 1988; Accepted March 23, 1989)     

A Single Equation to Quantify the Hierarchy in Plant Size Induced by Competition Within Monocultures

The following empirical model: Ra(i) = r(1+ln(w(i)/wm)Kn)(1–(w(i)/W))(1–(y/Y)) which is based on the logistic growth equation, is developedto describe the growth of differently sized individuals withinplant communities. The model is tested against extensive setsof carrot (Daucus carota L.) and red beet (Beta vulgaris L.)data and is shown to fit well. The model was used to predictindividual plant weights in independent data. The agreementsbetween observed and predicted weights were often close butsome systematic deviations did occur. Thus, a single equationdescribed most of the complex interactions that occurred withinmonocultures of annual crop plants. Carrot, Daucus carota L., red beet, Beta vulgaris L., model, growth, variation