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At least 343 records · Page 19Linked to original sources

PubMed1985

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization.

Mg:ATP-dependent H(+) pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Delta psi). DeltapH was measured as the decrease of absorbance of Acridine orange and Delta psi as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Delta pH and Delta psi generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Delta pH generation is not inhibited by NO(3) (-). These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H(+)-ATPases. Both intravesicular acidification and Delta psi generation are influenced by anions: Delta pH increases and Delta psi decreases following the sequence SO(4) (2-), Cl(-), Br(-), NO(3) (-). ATP-dependent H(+) pumping strictly requires Mg(2+). It is very specific for ATP (apparent K(m) 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Delta pH generation is inhibited by CuSO(4) and diethylstilbestrol as well as vanadate. Delta pH generation is specificially stimulated by K(+) (+ 80%) and to a lesser extent by Na(+) and choline (+28% and +14%, respectively). The characteristics of H(+) pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.

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PubMed1986

Localization of carbamoylphosphate synthetase and aspartate carbamoyltransferase in chloroplasts.

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed.

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PubMed1986

Effects of Dimethyl Sulfoxide and pH on Indoleacetic Acid-Induced Lateral Root Formation in the Radish Seedling Root.

Segments (2.5 cm) cut from 3-day-old seedling roots of radish (Raphanus sativus L. ;Scarlet Globe') were cultured in medium with or without indoleacetic acid (IAA). Lateral root primordia frequency, determined for the central centimeter of segments, was dependent on IAA concentration and on conditions affecting IAA uptake. Dimethyl sulfoxide treatment, or a relatively low medium pH, greatly enhanced the response to exogenous IAA. It was concluded that a permeation barrier exists between the external medium and the hormone responsive sites within the radish seedling root.

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PubMed1986

Phototropism in Hypocotyls of Radish : I. Isolation and Identification of Growth Inhibitors, cis- and trans-Raphanusanins and Raphanusamide, Involved in Phototropism of Radish Hypocotyls.

Three growth inhibitors which might be involved in phototropism of Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls, were isolated as crystalline forms from light-exposed radish seedlings and identified as cis- and trans-raphanusanins and 6-methoxy-2,3,4,5-tetrahydro-1,3-oxazepin-2-one (designated raphanusamide). The cis- and trans-raphanusanins inhibited growth of etiolated radish hypocotyls at concentrations higher than 1.5 micromolar, raphanusamide at concentrations higher than 20 micromolar.

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PubMed1986

Phototropism in Hypocotyls of Radish : II. Role of cis- and trans-Raphanusanins, and Raphanusamide in Phototropism of Radish Hypocotyls.

When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light (0.1 watt per square meter), the growth rate at the lighted side was strongly inhibited for the first 2 hours, while that at the shaded side showed no change. After 2.5 hours growth on the lighted side recovered gradually, while that on the shaded side was slightly inhibited. The neutral growth inhibitors, cis- and trans-raphanusanins and raphanusamide, were determined in the lighted and shaded sides from 1 hour before until 2 hours after the start of unilateral illumination. In the lighted side, cis- and trans-raphanusanins increased by 0.5 hour after the start of illumination, reached 3 to 3.5-fold greater concentrations than in the shaded side after 1 hour, and then decreased gradually. Raphanusamide increased in the lighted side to a 3-fold greater concentration than that in the shaded one 2 hours after the start of the illumination. Unilateral applications of cis- and trans-raphanusanins and raphanusamide suppressed the growth of the hypocotyl on the applied side more than that on the opposite one, causing the hypocotyls to bend towards the site of application. The data suggest that phototropic curvature in radish is caused by the light-induced synthesis of growth-inhibiting cis- and trans-raphanusanins, and raphanusamide at the site of illumination.

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PubMed1986

H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin.

The effect of fusicoccin on Mg:ATP-dependent H(+)-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the DeltapH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H(+)-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H(+)-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H(+)-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H(+)-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H(+)-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H(+)-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V(max) of the plasma membrane H(+)-ATPase rather than a decrease in its apparent K(m) for Mg:ATP.

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PubMed1987

Phototropism in Hypocotyls of Radish : III. Influence of Unilateral or Bilateral Illumination of Various Light Intensities on Phototropism and Distribution of cis- and trans-Raphanusanins and Raphanusamide.

When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light at 0.05, 0.1, or 1 watt per square meter, the suppression of the growth rate on the lighted side depended on the light intensity. The growth rate at the shaded side was only a little affected by the illumination at 0.05 and 0.1 watt per square meter but considerably suppressed by that at 1 watt per square meter. Upon a continuous unequal bilateral illumination, the growth rate was more strongly suppressed on the side of the higher intensity than on the side of the lower one, resulting in phototropic curvature toward the light source of the higher intensity. It was calculated from correlation analysis of light intensity and growth rate that, on an average, 6.9% of the irradiation applied to one side reached the opposite side. The amounts of cis- and trans-raphanusanins and raphanusamide in hypocotyls subjected to unilateral or unequal bilateral illumination increased much more at the side of the lighted or the higher intensity than at the opposite side. The present study demonstrates that phototropism in radish hypocotyl is correlated with and we conclude caused by a gradient of growth inhibition in the hypocotyl, depending on irradiation-induced amounts of cis- and trans-raphanusanins and raphanusamide.

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PubMed1987

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange.

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca(2+) upon addition of MgATP. MgATP-dependent Ca(2+) uptake co-migrates with the plasma membrane H(+)-ATPase on a sucrose gradient. Ca(2+) uptake is insensitive to oligomycin, inhibited by vanadate (IC(50) 40 micromolar) and erythrosin B (IC(50) 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca(2+) uptake is insensitive to protonophores. These results indicate that Ca(2+) transport in these microsomal vesicles is catalyzed by a Mg(2+)-dependent ATPase localized on the plasma membrane. Ca(2+) strongly reduces DeltapH generation by the plasma membrane H(+)-ATPase and increases MgATP-dependent membrane potential difference (Deltapsi) generation. These effects of Ca(2+) on DeltapH and Deltapsi generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca(2+) uptake into plasma membrane vesicles. The Ca(2+)-induced increase of Deltapsi is collapsed by permeant anions, which do not affect Ca(2+)-induced decrease of DeltapH generation by the plasma membrane H(+)-ATPase. The rate of decay of MgATP-dependent DeltapH, upon inhibition of the plasma membrane H(+)-ATPase, is accelerated by MgATP-dependent Ca(2+) uptake, indicating that the decrease of DeltapH generation induced by Ca(2+) reflects the efflux of H(+) coupled to Ca(2+) uptake into plasma membrane vesicles. It is therefore proposed that Ca(2+) transport at the plasma membrane is mediated by a Mg(2+)-dependent ATPase which catalyzes a nH(+)/Ca(2+) exchange.

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PubMed1987

Phototropism in Hypocotyls of Radish: IV. Flank Growth and Lateral Distribution of cis- and trans-Raphanusanins in the First Positive Phototropic Curvature.

The first positive phototropic curvature induced by a pulse of unilateral white irradiation (0.1 watt per square meter, 30 seconds) of etiolated and de-etiolated Sakurajima radish (Raphanus sativus var hortensis f. gigantissimus Makino) hypocotyls was analyzed in terms of differential growth and growth inhibitor contents of the hypocotyls. In both etiolated and de-etiolated hypocotyls, the growth rates at the lighted sides were suppressed whereas those at the shaded ones showed no change. De-etiolation treatment induced a larger difference between the growth rates at the lighted and shaded sides of the hypocotyls, resulting in a larger curvature of de-etiolated seedlings than of etiolated ones. The contents of growth inhibitors, cis- and trans-raphanusanins, increased in the lighted but not in the shaded halves of the hypocotyls of etiolated seedlings. In de-etiolated seedlings, the two inhibitors increased due to the de-etiolation treatment. When de-etiolated seedlings were exposed to a pulse of unilateral irradiation the level of the two inhibitors remained high along the lighted side for 1 h following the light pulse, whereas at the shaded side the contents of the inhibitors abruptly decreased upon transfer to the dark, the difference between their amounts in the lighted and shaded sides being larger than in etiolated seedlings. Another growth inhibitor, raphanusamide, did not respond to the phototropic stimulus, although its amounts increased by the de-etiolation treatment. These data suggest that cis- and trans-raphanusanins are involved in the first positive phototropic response of radish hypocotyls, and that de-etiolation magnifies the phototropic response through induction of a larger lateral gradient of the raphanusanins in the hypocotyls by the phototropic stimulus.

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PubMed1987

Effect of altered sink: source ratio on photosynthetic metabolism of source leaves.

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of (14)C detected in sugar phosphates and UDPglucose following (14)CO(2) supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO(2) fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans (14)C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO(2) fixation rate was constant for several days.

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PubMed1987

Generation of a membrane potential by electron transport in plasmalemma-enriched vesicles of cotton and radish.

Plasmalemma-enriched vesicles were isolated from cotton roots (Gossypium hirsutum L. cv Acala San Jose 2) and from germinating radish seeds (Raphanus sativa L. cv Tondo Rosso Quarantino). When 100 millimolar ascorbate was added to the grinding medium, the addition of ferricyanide to either preparation led to an inside positive membrane potential as measured by the accumulation of thiocyanate. It is suggested that electrons from ascorbate were being transported electrogenically across the membrane to ferricyanide, resulting in an accumulation of protons within the vesicle. The redox activity of the vesicles has some similarities to that occurring in intact cells, thus providing a simpler system to study the components and effects of transmembrane electron transport.

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PubMed1988

Experimental Studies on Lateral Root Formation in Radish Seedling Roots: II. Analysis of the Dose-Response to Exogenous Auxin.

Application of indoleacetic acid (IAA) and other auxins causes cultured radish (Raphanus sativus L. ;Scarlet Globe') seedling root segments to produce an increased frequency (FR, no. cm(-1)) of lateral roots (LR); in the absence of auxin, segments spontaneously form about 6 LR cm(-1). A dose-response study has revealed that the increase in FR follows a biphasic Michaelis-Menten relationship with the medium concentration of the undissociated form of IAA ([IAAH](m)). The fitted curve for phase I has a maximum response level (R(max)) of 5.2 LR per centimeter above the spontaneous FR; the [IAAH](m) giving half-maximal response (C(1/2)) is 21 nanomolar. For phase II, the values for R(max) and C(1/2) are 56 LR per centimeter and 11 micromolar, respectively. The response is variable in the transition concentration region between the two phases; in that region (but not, or much less commonly, at higher or lower [IAAH](m)), LR initiation may resume or continue after the first day. At and above 100 micromolar [IAAH](m), the roots are hyperstimulated and generally fail to respond. The developmental stage of LR formed in medium with very low [IAAH](m) (10 nanomolar) is enhanced compared to LR formed in medium lacking auxin; the stage is diminished at higher auxin levels, in inverse correlation with FR. Trends in the responses to NAA and IBA were similar, but NAA required only 0.03 times the dose of IAA, while IBA required 6 times the dose of IAA. These findings may be of use in a search for possible auxin receptors involved with LR initiation.

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PubMed1988

Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons.

Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)(+) RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS(1)) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS(2)) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS(1) sevenfold after 72 hours, and decreased rapidly that for GS(2) and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS(1) mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS(1) genes were activated, while those for GS(2) were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves.

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PubMed1989

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings.

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K(d) of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K(d) 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H(+)-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H(+)-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H(+)-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.

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PubMed1989

Inhibitory effect of gabaculine on 5-aminolevulinate dehydratase activity in radish seedlings.

We have compared the activity of 5-aminolevulinate dehydratase (5-ALAD) with the amount of protein detected by specific antibodies in rocket immunoelectrophoresis. Parallel kinetic evolutions of enzymic activity and amount of antigen were observed in radish (Raphanus sativus L.) cotyledons, both in complete darkness or under standard far red light involving phytochrome. However, the treatment of seedlings with gabaculine leads to an important decrease in enzymic activity, while the specific protein content is maintained. This inhibition is not overcome by the addition of glutamic acid, but by 5-aminolevulinic acid which points to a specific control of 5-ALAD activity by its substrate. As there is no discrepancy between the enzymic activity and the amount of antigen during the time course development of seedlings, this could confirm a coordinate cellular control between 5-aminolevulinic acid formation and 5-ALAD protein synthesis, both being amplified by the action of phytochrome.

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PubMed1989

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings.

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca(2+) uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca(2+)-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca(2+)-dependent ATPase is stimulated by the Ca(2+) ionophore A(23187) and is localized at the plasma membrane. Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca(2+) (half-maximal rate at about 70 nanomolar), and MgATP (K(m) 15-20 micromolar). Ca(2+) uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca(2+) uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca(2+)-ATPase described in this paper is the enzyme which drives active transport of Ca(2+) at the plasma membrane of higher plants.

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PubMed1989

Characterization of a Radish Nuclear Gene Expressed during Late Seed Maturation.

To study long-lived mRNAs stored in radish (Raphanus sativus) seed, we have selected clones from a dry seed cDNA library by differential screening. One of these clones, p8B6, whose mRNAs are abundant in the dry seed, was characterized. This clone hybridizes to an RNA class of approximately 600 nucleotides whose accumulation begins during the desiccation phase, reaches its maximum level in the dry seed, and is no longer detectable in 12 hour old seedlings. mRNAs hybrid-selected by p8B6 encode four polypeptides, but only two are compatible with the size class of RNAs detected by Northern analysis. Three of them have previously been identified as major ;early germination' polypeptides, and their synthesis has been shown to be induced prematurely in immature embryos by a desiccation treatment. The protein deduced from the p8B6 nucleotide sequence is 9 kilodaltons in size, highly hydrophilic, rich in Gly and Glu, and contains no Cys, Trp, and lie. The amino acid sequence shares good homology with that of two recently described seed proteins: a cotton late embryogenesis abundant protein and the wheat early methionine-labeled protein. Southern blot analysis suggests that the p8B6 sequence belongs to a very small gene family. The exact function of the product encoded by p8B6 remains to be determined.

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PubMed1990

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues.

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K(m) values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP(+) in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP(+) oxidoreductase. The effect of NaCl and MgCl(2) concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP(+) oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.

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