Polyamine Metabolism and Osmotic Stress : II. Improvement of Oat Protoplasts by an Inhibitor of Arginine Decarboxylase

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with dl-α-difluoromethylarginine (DFMA), a specific `suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.     

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.     

Inositol Trisphosphate Metabolism in Carrot (Daucus carota L.) Cells

The metabolism of exogenously added d-myo-[1-³H]inositol 1,4,5-trisphosphate (IP₃) has been examined in microsomal membrane and soluble fractions of carrot (Daucus carota L.) cells grown in suspension culture. When [³H]IP₃ was added to a microsomal membrane fraction, [³H]IP₂ was the primary metabolite consisting of approximately 83% of the total recovered [³H] by paper electrophoresis. [³H]IP was only 6% of the [³H] recovered, and 10% of the [³H]IP₃ was not further metabolized. In contrast, when [³H]IP₃ was added to the soluble fraction, approximately equal amounts of [³H]IP₂ and [³H]IP were recovered. Ca²⁺ (100 micromolar) tended to enhance IP₃ dephosphorylation but inhibited the IP₂ dephosphorylation in the soluble fraction by about 20%. MoO₄²⁻ (1 millimolar) inhibited the dephosphorylation of IP₃ by the microsomal fraction and the dephosphorylation of IP₂ by the soluble fraction. MoO₄²⁻, however, did not inhibit the dephosphorylation of IP₃ by the soluble fraction. Li⁺ (10 and 50 millimolar) had no effect on IP₃ metabolism in either the soluble or membrane fraction; however, Li⁺ (50 millimolar) inhibited IP₂ dephosphorylation in the soluble fraction about 25%.     

Rapid Methods for Determining Decarboxylase Activity: Arginine Decarboxylase

A rapid biochemical method for the determination of arginine decarboxylase (EC 4.1.1.19) activity has been developed for use in the routine clinical microbiology laboratory and correlated with similar procedures for ornithine and lysine decarboxylase (EC 4.1.1.18) systems. It is based on the detection of agmatine, the amine end product formed during growth on a synthetic medium containing arginine as the key amino acid. A modified diacetyl reagent is used to detect this amine after a differential butanol extraction of the cultures. This procedure can be used to detect this amine after a 1- to 4-hr incubation period (with the use of an initial concentrated inoculum) or with an overnight culture. Thus, both an indirect measurement based on the alkalinization of the medium and a lengthy incubation period were avoided. Parameters for optimal enzyme activity and the pertinent enzyme systems involved in arginine and agmatine catabolism are discussed in detail.     

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.     

Polyamine Biosynthetic Enzymes in Chlorella: Characterization of Ornithine and Arginine Decarboxylase

In a Chlorella culture grown asynchronously under autotrophicconditions, two biosynthetic enzymes of putrescine—ornithinedecarboxylase (ODC) and arginine decarboxylase (ADC)—weredetected. Both enzymes require pyridoxal phosphate and dithiothreitolfor their activity but differ in their optimal pH, the ionicstrength of their buffer, temperature of inactivation, and Km.In addition, L-canaline was found to inhibit the activity ofODC but not that of ADC. During the logarithmic phase of growth,ODC activity increased sharply, then decreased before the onsetof the stationary phase. ADC activity changed only slightlyduring growth. ³The work was performed in partial fulfillment of the requirementsfor the Ph.D. Thesis of E.C. (Received September 25, 1982; Accepted June 1, 1983)     

Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.)

Carbohydrate metabolism in Al-phosphate utilizing cells of carrot[designated as IPG, Koyama et al. (1992) Plant Cell Physiol.33: 171], which grow normally in Al-phosphate medium accompaniedby citrate excretion, was investigated. The excretion of citratewas strongly related to the availability of sucrose in medium,indicating that citrate excretion was severely limited by sucrosein medium. The ratio of the amount of carbon in the excretedcitrate to the consumed sucrose, was significantly higher inIPG cells than in wild-type cells. When 50% of the sucrose inthe medium was consumed, the ratio was 0.6% for the IPG cellsand 0.2% the wild-type cells. Under these conditions, IPG cellsshowed altered citrate synthesis metabolism, which resultedin increased citrate production. Specific activity of mitochondrialcitrate synthase was higher in IPG cells than in wild-type cells,whereas the activity of cytosolic NADP-specific isocitrate dehydrogenasewas lower in IPG cells than in wild-type cells. (Received August 27, 1998; Accepted February 21, 1999)     

Seasonal Changes of Polyamine Concentrations and Arginine Decarboxylase Activities in Leaves of 4 Ecotyes of Reeds

The seasonal changes of polyamine concentrations and arginine decarboxylase (ADC, EC 4.1.1. 1. 9)activities were investigated in the leaves of 4 ecotypes of reeds (Phragamites comrnunis Trinius)distributed over Hexi Corridor of Gansu province. The leaves of all ecotypes of reeds contained the same kind of polyamines and showed the same trend of decrement in total amuonts of potyamines with change of seasons. From May to September, the reeds which grow in arid and saline habitat maintained higher level of spermidine (Spd)and spermine (Spm)with no accumulation of putrescine (Put), resulting in low ratios of Put to other polyamine (Spd and Spm), whereas opposite results were observed in swamp reeds. These results indicate that the adaption of reeds to drought and salt stresses may correlate with Put synthesis via ADC pathway and the quick transformation of Put into Spd and Spm.     

石刁柏胚性细胞诱导过程中的内源激素和多胺含量变化

用高效液相色谱法分析石刁柏愈伤组织胚性细胞诱导过程中不同时期内源激素和多胺含量的结果表明,在胚性细胞诱导过程中,Put和GA3一直呈上升趋势,胚性细胞出现时,IAA、Put和GA3含量都达到最高水平,显示高含量的IAA以及高比例的Pu“（Spd＋Spm）可能有利于胚性细胞的形成。     

Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis

Tracer kinetic experiments were performed using [ureido-¹⁴C] citrulline, [1-¹⁴C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the ¹⁴C-solution and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the ¹⁴C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose ¹⁴C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO₂ up to 4 hours after introduction of label. All of the ¹⁴C in free and protein arginine could be accounted for in the C-6 position. Metabolism of ¹⁴C-ornithine resulted in ¹⁴C-incorporation into citrulline and free and protein arginine and the evolution of ¹⁴CO₂. Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of ¹⁴C. With the addition of 800 nanomoles unlabeled citrulline to the ¹⁴C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of ¹⁴C-citrulline increased 12-fold. The appearance of ¹⁴C in free arginine and protein arginine was also delayed. In both ¹⁴C-ornithine feedings all of the ¹⁴C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine → citrulline → argininosuccinate → arginine → protein arginine.     

Changes in ribosomal activity during incubation of Daucus carota root disks

Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg²⁺ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg²⁺, the ability to bind ³H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg²⁺ optima for poly U-directed ¹⁴C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg²⁺) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding ³H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks.     

Inhibitory Activity and Chemical Characterization of Daucus carota subsp. maximus Essential Oils

The essential oils (EOs) of green seeds from Daucus carota subsp. maximus growing wild in Pantelleria Island (Sicily, Italy) were characterized. EOs were extracted by steam distillation, examined for their inhibitory properties against food‐borne Gram‐positive and Gram‐negative bacteria and analyzed for the chemical composition by gas chromatography (GC) and mass spectrometry (MS). Undiluted EOs showed a large inhibition spectrum against Gram‐positive strains and also vs. Acinetobacter spp. and Stenotrophomonas maltophilia. The minimum inhibition concentration (MIC) was in the range 1.25 – 2.50 μl/ml for the most sensitive strains. The chemical analysis indicated that D. carota subsp. maximus EOs included 34 compounds (five monoterpene hydrocarbons, six oxygenated monoterpenes, 14 sesquiterpene hydrocarbons, four oxygenated sesquiterpenes, camphorene and four other compounds), accounting for 95.48% of the total oil, and that the major chemicals were carotol, β‐bisabolene, and isoelemicin.     

Changes in Endogenous Amino Acid Compositions during Somatic Embryogenesis in Daucus carota L.

Changes in amino acid compositions of carrot cells culturedin three different media were examined in relation to somaticembryogenesis. The total amount of amino acids in the 80% ethanol-insolublefractions increased rapidly during cell proliferation and globularembryo formation. The individual proportions of the amino acidsin these fractions remained fairly constant irrespective ofthe development of the somatic embryos. The total amount ofamino acids in the 80% ethanol-soluble fractions varied dependingon the medium used. Continuously applied -alanine did not accumulated in the cells,which suggests that it was transformed to other amino acidsduring culture. A large amount of aminobutyric acid was detectedthroughout the culture period. Glutamic acid and glutamine accumulatedin the carrot cells during embryo formation and maturation.Changes in the compositions and amounts of the endogenous aminoacids present during embryo formation are discussed. (Received April 28, 1983; Accepted October 5, 1983)     

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

We have shown previously that inositol-1,4,5-trisphosphate (IP₃) stimulates an efflux of ⁴⁵Ca²⁺ from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP₃ might serve as a second messenger for the mobilization of intracellular Ca²⁺ in higher plant cells. To determine whether or not IP₃ and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-³H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [³H]inositol metabolites coeluted with inositol bisphosphate (IP₂) and IP₃ when separated by anion exchange chromatography. However, we could not detect IP₂ or IP₃ when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP₂ and IP₃, were present in these cells. Thus, [³H] inositol metabolites other than IP₂ and IP₃ had coeluted on the anion exchange columns. The data indicate that either IP₃ is rapidly metabolized or that it is not present at a detectable level in the carrot cells.     

IAA Metabolism in Embryogenic and Non-Embryogenic Carrot Cells

Carrot somatic embryos can readily be induced from embryogeniccells transferred from auxin-containing medium to auxin-freemedium, but not from transferred non-embryogenic cells. It iswell-known that IAA, a natural auxin, plays important rolesin many physiological responses including somatic embryogenesis,but, there is no report of the IAA metabolism in embryogenicand non-embryogenic cells. Therefore, we examined IAA metabolismin embryogenic and nonembryogenic carrot cells. In this paper the IAA metabolism in embryogenic cells and non-embryogeniccells is described. The induction of IAAsp formation was clarifiedin both cells. On the other hand, in non-embryogenic cells,an unknown metabolite was detected and identified as oxindole-3-acetylasparticacid (oxIAAsp). OxIAAsp formation may be induced to eliminateexcess auxin. Furthermore, endogenous IAA contents in both cellswere quantified and the relationship between somatic embryogenesisand IAA metabolism is discussed. (Received May 2, 1994; Accepted August 30, 1994)     

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.     

The Fine Structure and Development of Plastids in Cultured Cells of Daucus carota

An investigation has been made of the plastids in cells of asepticallycultured carrot explants using a range of fixatives and stainingprocedures. Attention was focused upon the plastids as theyoccur in quiescent cells of the carrot root, in explants exposedto a basal medium which does not cause rapid growth, and inexplants which were subject, in both the light and the dark,to stimuli that cause cell division. These cell-division stimuliwere supplied by the use of the liquid endosperm of the coconut,which also induces free cells to behave totipotently and togrow like zygotes. Fully differentiated and functional chloroplastsare formed when cells are exposed to the coconut-milk-supplementedmedium in the light. By contrast, the development of plastidsis arrested when the cells are grown in the dark at a stagewhich, although more complex than the state of the plastidsin the storage root, nevertheless falls far short of the chloroplastwhich develops in the light. Plastids in dark-grown cells aremembrane-bounded and, from the inner membrane, imaginationspenetrate the densely granular 8troma. Two bodies, or organelles,characterize the plastid of dark-grown cells. One consists ofa membrane-bounded, spherical structure the content of whichis very electron-dense after glutaraldehyde fixation but muchless so after treatment with osmium tetroxide. Because thisbody is progressively and demonstrably transformed in the lightinto thylakoids and grana it is named the prethylakoidal body.The other plastid inclusion to which attention is directed isa spherical entity, not visibly membrane-bounded, but whichhas lipid droplets disposed on its surface; this is termed theglobular centre. The materials for the structure of the chloroplastlamellae and grana accumulate in the dark in the prethylakoidalbody; in its metamorphosis in the light this structure formslong continuous loops of spirally twisting thylakoids which,as they close down upon each other, form discrete grana. Asthe thylakoids enlarge and develop, their association with thelipid droplets of the globular centre suggests that this bodyplays an integral part in chloroplast development. Special attentionis given to the details of thylakoidal structure, in so faras these may be revealed in fixed material, and the resultsare compared with other relevant studies. Comparisons of themembranes at the surfaces of thylakoids and at the cell surface(plasma-lemma), as these are affected by different preparativeregimes, have been made and interpreted. Thus plastids of carrotrespond to the complex of factors that stimulate the growthof the cells and, in doing so, they form structures (the prethylakoidalbody and the globular centre) in the dark which, under the influenceof the light, metamorphose into the system of thylakoids andgrana. Further questions are now posed for subsequent investigations.     

Endosperm and Embryo Development in Daucus carota L.

Maximum carrot seed dry weight and maximum endosperm volumewere reached about 35 d after anthesis, although at this timethe endosperm was still soft, the pericarp green and less than50% of the seeds were viable. Fully viable, ripe seeds werenot produced until 44 d later. Seventy per cent of the increasein endosperm volume was due to an increase in cell number, whichceased 35 d after anthesis. The increase in embryo volume wasslower and was due to an increase in both cell number and cellvolume which continued until 49 d after anthesis. At maturitythe embryo was the equivalent of between 2% and 3% of the endospermvolume. The relationship between embryo length and cell number per embryowas unaffected by seed crop plant density, seed crop harvestdate and position of the seed on the mother plant but it wasaffected by the year of seed production, possibly due to differencesin temperature during the period of seed growth. Key words: Endosperm, Embryo, Carrot, Development     

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     

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.