Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings XI. Inhibition of RNA Synthesis by 4-Thiouridine Nucleotides during Germination of Radish Seeds

The effects of 4-thiouridine and its metabolites on RNA synthesisin radish (Raphanus sativus) cotyledons were investigated. The4-thiouridine nucleotides, 4-thioUTP and 4-thioUDP, were foundto inhibit DNA-dependent RNA polymerase activities of isolatednuclei and of chloroplasts from radish cotyledons. These inhibitorsappeared to compete with UTP for its binding to RNA polymerase.Neither 4-thiouridine nor 4-thioUMP inhibited RNA polymeraseactivities. Reduced RNA accumulation accompanied by the inhibition of protochlorophyllideaccumulation was observed in cotyledons of dark-grown radishseedlings germinated and grown with 4-thiouridine. On the otherhand, 4-thiouridine had no effect on chloroplast developmentin the control seedlings germinated and grown without 4-thiouridine.These results suggest that the inhibition of chloroplast developmentby 4-thiouridine may in part be due to the inhibition of RNAsynthesis by 4-thiouridine nucleotide during germination, resultingin inhibition of etioplast development. (Received December 9, 1985; Accepted June 26, 1986)     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings V. Protein Synthesis in Vivo

Incorporation of ¹⁴C-amino acids into proteins in radish cotyledonswas suppressed by 4-thiouridine (4SU) culture. The inhibitoryeffect of 4SU was similar to that of chloramphenicol. 4SU culturedid not reduce the content of ferredoxin (Fd) and the labelinginto Fd significantly, but it did decrease the content and thesynthesis of ribulose bisphosphate carboxylase (RuBPCase). Thesynthesis of thylakoid chlorophyll-proteins I and II also wasinhibited by 4SU culture. In 4SU-cultured seedlings, the ratioof labeling into the large and small subunits of RuBPCase andthat into the two chlorophyll-proteins were the same as thosein the controls grown without 4SU. (Received September 29, 1980; Accepted January 27, 1981)     

Effect of 4-Thiouridine on Chloroplast Development

The methoxymethyl group of the 6-methoxy-2-methoxymethyl-3-(3,4-methylenedioxyphenyl)-1,4-benzodioxan-7-yl moiety of insecticidallignans of Phryma was modified with sereral alkyl groups to evaluate the effect on activity of the substituents. The assay results make it evident that this activity was significantly modified by the chain length or bulkiness of the alkyl groups and that the oxygen atom of the methoxymethyl group was fairly important for enhancing the activity.     

Development of Chloroplast in Dark-Grown Wheat Seedlings Irradiated at Elevated Temperature

Elevated temperature inhibited the accumulation of chlorophyll and photosynthetic proteins, and the development of photochemical activity, however, carotenoids continued to accumulate. Signal transduction pathway involved in protochlorophyllide oxidoreductase was unaffected by elevated temperature of 38°C. Two-dimensional gel electrophoresis of stroma proteins showed similar patterns in the dark-grown seedlings and seedlings irradiated at elevated temperature, although some low molecular mass proteins accumulated at 38°C. In contrast, seedlings irradiated at 25°C showed complex pattern of proteins. Hence the development of chloroplast and its associated functions during irradiation of etiolated seedlings are inhibited by elevated temperature.     

Structural Changes in Radish Seedlings Exposed to Cadmium

Radish (Raphanus sativus L. cv. Redondo Vermelho) seedlings were analysed by light and scanning electron microscopy to characterize the structural changes caused by the exposure to 0.5 or 1.0 mM cadmium chloride for 24, 48 and 72 h. The analyses showed changes in the anatomical and morphological characteristics of roots, stems and leaves of two-week-old seedlings. In all tissues, pressure potential was decreased. Premature death with the disintegration of the epidermis and an increase in the number of root hairs was observed in roots exposed to Cd. The stem of seedlings exposed to Cd exhibited more cells layers in the cambial region. The main effects observed in leaves in response to Cd were stomatal closure, lack of cell wall thickening and alterations in the shape of the chloroplasts. It is suggested that the structural changes observed in seedlings treated with Cd were mainly caused by a Cd-induced decrease in water uptake.     

The Reactivity of 4-Thiouridine with Peroxidase and Superoxide Radicals

Rice leaf slices stimulated with blast fungus hyphal component reduced nitroblue tetrazolium in a damped oscillatory profile with relaxing half wavelength in a medium containing glucose, when the respective rate of reduction was plotted against the function of time after the application of blast fungus hyphal component. In the presence of 110μm FAD and glucose, the wave number of the reduction profile increased 4- to 5-fold when compared to that in the absence of exogenous FAD. Exogenous FAD in the increasing concentration of 70 to 110 μm, which was added in the presence of glucose, gave a positive heterotropic-like response upon the reduction of nitroblue tetrazolium with rice leaf slices which were press-injured and stimulated. Exogenous pyrroloquinoline quinone in the increasing concentration of 10^?3 to 10^?1 μm, which was added in the presence of glucose, gave an inhibition upon the reduction. From sediment of the homogenate of stimulated rice leaf slices, the nitroblue tetrazolium reducing redox-enzyme system was solubilized by Triton X-100 and was electrophoretically isolated in a sharp blue band on a polyacrylamide slab gel containing Triton X-100, when the electrophoresed gel was stained by nitroblue tetrazolium or Coomassie brilliant blue. In the solubilized solution, the presence of b-type cytochrome was observed by the oxidation-reduction difference spectrum.     

Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin

The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca²⁺ concentration. Stimulation was marked at low, nonsaturating Ca²⁺ concentrations and decreased increasing Ca²⁺, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca²⁺. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca²⁺, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.     

The Metabolism and Translocation of Zeatin in Intact Radish Seedlings

After the roots of intact radish seedlings had taken up [³H]zeatinfor 1 h, the seedlings were transferred to nutrient lackingzeatin and extracted at intervals. After 23 h in the absenceof zeatin, 6 per cent of the radioactivity extracted per seedlingwas recovered from the de-ribbed cotyledon laminae, 4 per centfrom the hypocotyls, and 87 per cent from the roots. Per unitweight of tissue, the radioactivity extracted from the rootwas about 40 times that recovered from any other region. Zeatin was rapidly metabolized by the root tissue, and 4 to9 h after transfer of the seedlings to nutrient lacking zeatin,accounted for a negligible proportion of the radioactivity.Initially zeatin riboside 5'-monophosphate was the principalroot metabolite, but after 9 h, 7-glucosylzeatin (raphanatin)was the dominant metabolite. Conversion of zeatin to dihydrozeatinwas not detected. Raphanatin was also the major metabolite inthe cotyledon laminae where some free zeatin was detectable.The principal metabolites in hypocotyl extracts were AMP andzeatin riboside 5'-monophosphate but zeatin riboside was theonly significant source of radioactivity in the xylem sap. When [³H]zeatin was applied directly to cotyledon laminae, 99per cent of the radioactivity was localized in the treated laminae;however traces of zeatin were detected in the roots. In radish seedlings, zeatin riboside appears to be the translocationalform of zeatin, while raphanatin may be a storage form.     

Effect of 4-Thiouridine on Photosystems of a Higher Plant

Effect of 4-thiouridine, which was proved to inhibit selectively and “light-reversibly” the synthesis of chloroplast ribosomal RNAs in radish cotyledons, on the photo-induced development of photosystem I, II and a complete electron transport chain was investigated with plastids obtained from 4-thiouridine treated dark-grown radish cotyledons after various times of development in the light. It was demonstrated that the 4-thioridine treated chloroplasts showed a higher activity of photoreduction than the control untreated chloroplasts in every system on a chlorophyll basis during the development after 24 hr illumination. This specific activity decreased in both chloroplasts, as the chloroplasts matured with the time of illumination. The activity per g of fresh cotyledons treated with 4-thiouridine, especially in the early stage of development, was lower than that of ones untreated with the drug because total chlorophyll content was poor, but the activity of the former was enhanced with the increase of total chlorophyll content upon illumination while the activity of the latter decreased on 24 hr illumination. Moreover, Hill reaction measurements showed that 4-thiouridine treated chloroplasts were saturated at lower light intensity than untreated ones inspite of the same content of chlorophyll in both the chloroplasts: photoreduction of NADP⁺ was saturated at 3000 lux for the former and at 5000 lux for the latter. Based upon these results, specific development of the chloroplast is discussed.     

Storage Organ Development in Radish (Raphanus sativus L.). 1. A Comparision of Development in Seedlings and Rooted Cuttings of Two Contrasting Varieties

Development was compared in two contrasting varieties of radishgrown in nutrient solution. Differences in shape of the long,tapering radish of Long White Icicle and the smaller, roundCherry Belle were associated with differences in the growthof the upper part of the tap root. Thickening was the resultof , cambial activity and transverse scctions through the upperroot showed differences in both cell number and a l l size.Long White Icicle contained more cells in the secondary xylemand phloem/secondary cortex than Cherry Belle, and the non-lignifiedxylem parenchyma cells were also larger in Icicle. The upperpart of the tap root also elongated in Icicle but not in CherryBelle, and this was entirely due to an increase in cell numberin the longitudinal plane. Removing the distal part of the root system had little effecton storage organ size unless most of the root system was removed.Adventitious roots on seedling and shoot-tip cuttings of bothvarieties were capable of thickening and the single root developingon shoot-tip cuttings of Cherry Belle achieved a diameter greaterthan that of the radicle of the intact plant. Rooted cotyledonsof Cherry Belle cxrasionally produced small storage organs. Raphanus sativus, radish storage organ, root thickening, cambial activity     

Raphanusol B: A Growth Inhibitor of Light-grown Radish Seedlings

A growth inhibitor, for which the name raphanusol B is proposed,was isolated in crystalline form from light-grown Sakurajimaradish seedlings and has been shown to be 1-sinapoylglucoseby spectrometric analysis. Raphanusol B inhibited the growthof intact and excised hypocotyls of etiolated radish seedlings.The raphanusol B content of the radish seedlings increased greatlyunder red light, but decreased in the dark. ¹Present address: Department of Chemistry, Faculty of Science,Kagoshima University, Korimoto 1, Kagoshima 890, Japan. (Received October 6, 1980; Accepted December 6, 1980)     

Studies of Chloroplast Development in Euglena: XIII. Variation of Ultraviolet Sensitivity with Extent of Chloroplast Development

Ultraviolet (UV) inactivation of green colony-forming ability of several different types of Euglena gracilis var. bacillaris was studied. The observed target numbers are not widely different, while the doses required to produce a single inactivation event (D(o)) vary with the type of cell used. In dark-grown cells adapting to the light in resting medium and in an X-ray-induced mutant, D(o) is proportional to the chlorophyll content of the cells. However, in hyperdeveloped cells which contain abnormally high amounts of chlorophyll, the correlation does not hold, suggesting that it is not chlorophyll per se which is responsible for the differences observed. D(o)'s of colony-forming ability (viability) of light-grown and dark-grown cells are found to differ by the same factor as those of green colony-forming ability. Stationary phase and exponential phase cells show a small difference in D(o) with no obvious difference in target multiplicity. The multiplicity of the various target curves has been re-evaluated by computer and found to be between 30 and 40.     

Chloroplast and Cytoplasmic Enzymes: VI. Pea Leaf 3-Phosphoglycerate Kinases

Pea (Pisum sativum) leaf chloroplastic and cytoplasmic 3-phosphoglycerate kinases (ATP: d-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) have similar Michaelis constants for ATP, 0.7 and 0.55 mm, for ADP, 0.18 and 0.22, and for 3-P-glycerate, 0.59 and 0.54 mm at low substrate concentrations, and 1.6 and 1.25 mm at high substrate concentrations. Both enzymes are inhibited by ADP and AMP in the ATP-utilizing direction and by ATP and AMP in the ATP-generating direction and are controlled by energy charge. Apparently, whether the cytoplasmic and chloroplastic kinases in the plant cell will participate in the reductive pentose phosphate cycle and gluconeogenesis or in glycolysis will be determined by the environment in the cell compartment and not by the differential properties of the enzymes themselves.     

Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

ThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. In Escherichia coli and Salmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, in Bacillus subtilis and most species from the Firmicutes phylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis of B. subtilis thiI and the adjacent gene, nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments in B. subtilis indicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine. In vitro synthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain. In vivo complementation studies in E. coli IscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal that B. subtilis NifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Plasma Membrane Ca-ATPase of Radish Seedlings : I. Biochemical Characterization Using ITP as a Substrate

In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The majority of the ITPase activity of plasma membrane was Ca(2+)-dependent. The Ca(2+)-dependent ITPase activity was Mg(2+)-dependent and was stimulated by the calcium ionophore A23187. It was inhibited by erythrosin B (concentration giving 50% inhibition, 50 nanomolar) and by vanadate (concentration giving 50% inhibition, 3 micromolar) and displayed a broad pH optimum around pH 7.2 to 7.5. Both the hydrolytic and the transport activity of the plasma membrane Ca-ATPase were half-saturated by Ca(2+) in the micromolar concentration range. No major effect of EGTA on the saturation kinetics of the enzyme was observed. The affinity of the plasma membrane Ca-ATPase for Ca(2+) was about fourfold higher at pH 7.5 than at pH 6.9. The Ca(2+)-dependent ITPase activity was stimulated about twofold by polyoxyethylene 20 cetyl ether, although it was inhibited by Triton X-100 and by lysolecithin.