Uptake of uridine by a long-day duckweed, Lemna gibba G3

Uptake of uridine by a long-day duckweed, Lemna gibba G₃ wasexamined. Km and Vmax for uptake were in the range of 1 to 2x10^–5 M and of 5 to 10 x10^–8 moles/g fresh weight/2hr, respectively. Uptake rate depended on temperature, and theoptimum pH was 5.0. Uridine uptake was competitively inhibitedby some compounds structurally analogous to uridine. However,the activity of uridine kinase was not affected by these compounds,except for cytidine. Uridine uptake was inhibited by metabolicinhibitors, in which uridine taken up was left unconverted toother forms, especially in the presence of DNP. These resultssuggest that uridine was taken up into the duckweed celb bya specific transport system and immediately phosphorylated byuridine kinase. Phosphorylation of uridine was not associatedwith the uridine transport reaction. (Received November 15, 1976; )     

MORPHOGENESIS IN SPIEODELA OLIGORRHIZA: EFFECTS OF PYRIMIDINE BASE ANALOGUES ON INITIATION, ELONGATION AND DIFFERENTIATION OF FRONDS

The effect of four pyrimidine base analogues and their antidoteson S. oligorrhiza was studied. FUdR stopped cell division at concentrations of 4 10^–7M and higher. This effect could be nullified by the additionof 4 10^–6 M thymidine. Neither uridine nor uracil hadan antidotal effect on FUdR. FU (8 10^–6 M or higher concentration) affected celldivision, frond elongation and differentiation, and could notbe counteracted by either thymidine or uracil. TU (8 10^–4 M) rather specifically inhibited differentiationof frond tissues, while not preventing cell division or theinitiation of new generations. Uracil and uridine at about equimolarconcentrations completely counteracted the TU effect. AzU (10^–3 M) suppressed cell division, frond elongationand frond differentiation. When thymidine (10^–3 M) wasadded simultaneously with AzU only cell elongation and differentiationof fronds were inhibited, but cell division proceeded. 10^–3M uracil (but not uridine) counteracted all effects of AzU. ¹ Based on a portion of the senior author's Ph.D. Thesis.     

Induction of DNA synthesis and callus formation from tuber tissue of Jerusalem artichoke by 2,4-dichlorophenoxyacetic acid

Cellus induction was observed from Jerusalem artichoke tubertissue on a synthetic medium containing 2,4-D at 10^–6,10^–5 (optimum conc.) and 10^–4 M. The first DNA synthesis(thymidine incorporation) was observed only at 2,4-D concentrationsof 10^–5 to 10^–4M. In 10^–5 M 2,4-D treatedtissue, DNA synthesis increased after a 20 hr lag and reacheda maximum at 36 hr, after which it decreased. Actinomycin Dand 8-aza-guanine; inhibitors of RNA synthesis, inhibited DNAsynthesis completely. 2,4-D caused the characteristic changesin RNA and protein syntheses. In comparison with the control,RNA and protein syntheses were first repressed then inducedbefore the peak of DNA synthesis. Treatment with cycloheximide(10^–4M) for one hour before inoculation inhibited proteinsynthesis completely for 12 hr; consequently DNA synthesis wasalso delayed. The results suggest that RNA and protein synthesesneeded for callus induction are regulated by 2,4-D in the firstDNA synthesis. (Received July 19, 1973; )     

Studies on CN--insensitive respiration in plant roots III. Induction of CN--insensitive respiration with low concentrations of respiratory inhibitors

Induction of CN^–-insensitive respiration with low concentrationsof respiratory inhibitors was studied. If roots were treatedwith 10^–3 M CN^– for 96 hr, the plants died, whilethose treated with 10^–4 M CN^– showed healthy growth. O₂ uptake in untreated rice and wheat roots showed a negativeresponse to 10^–2 M CN^– to a considerable extent.On the other hand, pretreatment with 10^–4 M CN^–for more than 6 hr did not greatly affect respiratory rate,but made respiration insensitive to 10^–2 M CN^–.A similar induction of CN^–-insensitivity was also broughtabout with 10^–4 and 10^–3 M H₂S and 10^–4 MNaN₃. (Received July 6, 1971; )     

A simple method for the isolation of intact mesophyll protoplasts from cereal plants

Intact mesophyll protoplasts from cereal plants were easilyprepared by incubating leaves with the abaxial epidermis peeledoff at 20–25?C for 2–3 hr in 0.6 M mannitol containing1% cellulase at pH 5.6. From one gram (fresh weight) of leaves1.5–6?10⁶ protoplasts, more than 90% of which were morphologicallyintact, could be obtained. Protoplasts isolated from wheat,oat, corn and barley were efficiently infected with brome mosaicvirus (BMV), and supported viral multiplication. (Received June 21, 1977; )     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Microelectrophoretic and 9-Aminoacridine Fluorescence Study of the Surface Electrical Properties of Suspension Cultured Catharanthus roseus Cells and Isolated Protoplasts: Effects of Abscisic Acid Treatment

The effects of abscisic acid (ABA) treatments on the surfaceelectrical properties of cells and isolated protoplasts fromCatharanthus roseus cell suspension cultures were studied byelectrophoretic mobility and 9-aminoacridine (9AA) fluorescencemeasurements. The surface charge densities of the cells andprotoplasts estimated from electrokinetic data were –0.064Cm^–2and –0.048 C m^–2 respectively. These values wereclose to that estimated by 9AA fluorescence technique i.e.,–0.053 Cm^–2 for the cells and –0.041 Cm^–2for the isolated protoplasts accordingly. The net negative surfacecharge density decreased after application of 10 µM and50 µM ABA in both cells and protoplats, the more pronouncedeffect being observed at 10 µM ABA. When 100 µMABA was supplemented to the cell suspension culture the oppositeeffect was observed. The average charge density increased to–0.074 C m^–2 for the cells, and to –0.055C m^–2 for protoplasts, as revealed from the 9AA measurements.The results are discussed in terms of specific concentrationdependent ABA-induced alterations of the electrostatic propertiesof cell and protoplast membranes. (Received December 12, 1994; Accepted April 3, 1995)     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )     

Stimulation of the Uptake of Sorbitol into Vacuoles from Apple Fruit Flesh by Abscisic Add and into Protoplasts by Indoleacetic Acid

The uptake of sorbitol into vacuoles from immature flesh ofapple fruit (Maluspumila Mill, var domestica Schneid.) was facilitatedby 10^–6 M ABA, while such uptake into protoplasts wasnot stimulated. By contrast, the application of 10^–5 MIAA facilitated uptake of sorbitol into protoplasts but didnot significantly into vacuoles. (Received July 17, 1990; Accepted December 25, 1990)     

Metabolism of Pyrimidines in Protoplasts from Cultured Catharanthus roseus Cells

The metabolism of [2-¹⁴C]thymine, [2-¹⁴C]thymidine, [2-¹⁴C]uraciland [¹⁴C]uridine was investigated in protoplasts obtained fromsuspension cultures of Catharanthus roseus. Most of the exogenouslysupplied thymine, thymidine and uracil was degraded, and salvageof these pyrimidines accounted for 5–36 per cent of thetotal amount of ¹⁴C-labelled precursors which was metabolized.However, more than 80 per cent of the labelled uridine was utilizedfor the biosynthesis of nucleotides and nucleic acids, and therest was degraded. In contrast to the results from protoplastsof sugar cane cells in suspension culture, the data indicatethat protoplasts possess a pathway for the degradation of pyrimidines,and that the overall metabolism of these pyrimidines in protoplastsis very similar to the metabolism in the intact cells. Catharanthus roseus, madagascar periwinkle, protoplasts, pyrimidine metabolism     

Studies on CN--insensitive respiration in plant roots V. Changes in glucose metabolism caused by 10-4 M CN- pretreatment

Metabolic changes accompanied by the induction of CN^–-insensitiverespiration in plant roots were investigated. Glucose-U-¹⁴Cwas administrated to untreated and 10^–4M CN^– pretreatedrice roots in the presence or absence of 10^–2M CN^–.¹⁴C incorporation into malate increased and that into citratedecreased remarkably with the 10^–4M CN^–pretreatment.10^–2M CN^– generally suppressed the incorporationof ¹⁴C into organic acids. However, the ¹⁴C incorporation intoaspartic and glutamic acids was not reduced, and that into alanineincreased greatly in the presence of 10^–2 M CN^–. (Received October 16, 1972; )     

Effect of Metabolic Inhibitors on the Formation of Cell Walls in a Green Alga, Micrasterias crux-melitensis

The effects of cytochalasin B, N-ethylmaleimide (NEM), colchicine,vinblastine and cycloheximide on the formation of birefringentcell wall layers were studied. Birefringent layers accumulatedoutside the plasma membrane of daughter semicells when cellswere cultured in a 0.16 M mannitol solution without any inhibitors.In cells treated with 2 x 10^–5 M cytochalasin B, 3 x 10^–5M NEM, 10^–4 M vinblastine or 10^–5 M cycloheximidefor 24 hr, birefringent layers were not observed outside theplasma membrane, but were present in cells treated with 10^–2M colchicine. The possibility is discussed that substances necessaryfor wall synthesis could be transported from the cytoplasm tothe outside of the plasma membrane by a system associated withmicrofilaments, microtubules and myosin-like structures. (Received June 26, 1981; Accepted September 24, 1981)     

Photosystem I and photophosphorylation-dependent leucine incorporation in the blue-green alga Anacystis nidulans

L-Leucine uptake and incorporation in the blue-green alga Anacystisnidulans were measured during illumination with monochromaticlight of 630 and 717 nm. With near as well as far red light,an enhanced uptake of ¹⁴C-L-leucine was observed. In far redlight, the leucine uptake depended on light intensity and pHvalue. After the first few minutes, the uptake remained constantfor more than one hour. The rate of uptake in light was thesame in air as in nitrogen. The incorporation of ¹⁴C-leucinein the soluble fraction decreased in the presence of chloramphenicolwhich prevents protein synthesis. In far red light, its incorporationwas insensitive to DCMU (5 ? 10^–6 M) but was depressedby uncouplers like CCCP or desaspidin. These effects are takenas evidence that leucine incorporation under the conditionsused is dependent on photosystem I reactions and cyclic photophosphorylation.DBMIB and KCN in high concentrations decrease the leucine incorporationin far red light and indicate that plastoquinone and plastocyaninare members of the cyclic electron flow also in intact cellsof Anacystis. Antimycin A has no inhibitory effect. The inhibitionby other less specific inhibitors like salicylaldoxime, desaspidinand DSPD is discussed. (Received August 19, 1978; )     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

A study on senescence in tobacco leaf disks I. Inhibition by benzylaminopurine of decrease in protein level

The drop in protein level as an index of senescence in tobaccoleaf disks was examined in the dark in the presence of BA and/orinhibitors of protein synthesis. Cycloheximide at 10^–6–10^–5M and 10^–5 g/ml actinomycin-D accelerated the senescenceand interfered with the anti-senescence action of 10^–6M BA. Chloramphenicol (10^–8– 10^–4 M) and puromycin(10^–8–10^–4M) did not modify the senescenceor the action of BA. Cycloheximide was more effective at earlierstages of the dark culture of the disks. The rate of ¹⁴C-leucineincorporation into the protein fraction was increased by BAand decreased with senescence, and this drop was removed byBA. The incorporation was also suppressed by cycloheximide equallyin the presence and absence of BA. The drop in labeled proteinof the disks during the chasing period was retarded by not onlyBA but cycloheximide also. The conclusion reached, based onthese and relevant findings, was that BA affects the anti-senescenceaction by promoting synthesis and at the same time inhibitingdegradation of protein. (Received December 2, 1974; )     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

The Generation of Non-Spherical Oat Protoplasts Indicates that Hyperpolarization is both Necessary and Sufficient for Stimulating Membrane Incorporation into the Plasma Membrane

We have devised conditions which produced isolated protoplastsof non-spherical shape and which, therefore, affected the mechanismsthat control the exchange of membrane material between the plasmamembrane and an intracellular membrane reservoir. Non-sphericalprotoplasts of Avena sativa were obtained if protoplasts weretreated with hypertonic shock in the presence of 1.0 mol m^–3LaCl₃ at pH 8.3. This indicated that their ability to removeplasma membrane material via endocytotic vesiculation was suppressed.Non-spherical protoplasts were obtained under isotonic conditionsif protoplasts were incubated with 1.0 mol m^-3 LaCl₃ at pH 8.3and the proton carrier CCCP (12 mmol m^–3) was added. Thenon-spherical protoplasts had intact membranes as judged bystaining with fluorescein diacetate. The loss of the sphericalshape was reversible. On addition of EDTA protoplasts resphericulatedimmediately. Incubation in isotonic solution at pH 8.3 containingeither only 1.0 mol m^–3 LaCl₃ or only CCCP did not influencethe protoplast shape. We conclude that the membrane hyperpolarizationinduced by CCCP at high pH acted to stimulate the incorporationof membrane material into the plasma membrane and, subsequently,produced nonspherical protoplasts if the removal of membranematerial was simultaneously suppressed. This demonstrates thatmembrane incorporation and removal are two largely independentprocesses.     

Effects of Light and Inhibitors of Photosynthesis and Respiration on the Multiplication of Tobacco Mosaic Virus in Tobacco Protoplasts

The multiplication rate of tobacco mosaic virus (TMV) in tobaccoprotoplasts in light was several times than in the dark. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea(DCMU) at 10^–5M completely antagonized this illuminationeffect. KCN at 10^–4 M and antimycin A at 10^–5 M,which prevented the protoplasts from surviving in the dark,did not block TMV multiplication in light. Inhibitor experimentsshowed that photosynthesis and respiration were indirectly associatedwith the TMV multiplication. Either of them was found to benecessary for TMV multiplication but neither was indispensable.They play complementary roles in the supply of energy and materialsrequired for virus production. (Received August 2, 1984; Accepted July 9, 1985)     

Role of Polysaccharide Synthesis in Elongation Growth and Cell Wall Loosening in Intact Rice Coleoptiles

2,6-Dichlorobenzonitrile (DCB) inhibited only increases in levelsof the cellulosic polysac-charides while monensin and galactoseinhibited increases in levels of both the cellulosic and thematrix polysaccharides in intact rice coleoptiles that weresubmerged in water. Elongation growth of rice coleoptiles wassuppressed by DCB at 10^–6 M, by monensin at 10^–7M, and by galactose at 3 ? 10^–3 M and above. Thus, thesynthesis of both the cellulosic and the matrix polysaccharidesis essential for the elongation of intact rice coleoptiles.These inhibitors increased the minimum stress-relaxation timeand the relaxation rate and they decreased the mechanical extensibilityof the cell wall, indicating that they inhibited cell wall loosening.The concentrations of the inhibitors required for inhibitionof cell wall loosening were higher than those for suppressionof elongation. The data suggest that polysaccharides synthesisplays two roles in elongation. It keeps the cell wall in a "loosened"condition by producing new extensible cell walls, while itsother role is probably related to the fixation or extensionof polymers already present in the cell wall. (Received November 15, 1990; Accepted May 23, 1991)