Interactions between the Nucleus and Cytoplasmic Organelles during the Cell Cycle of Euglena gracilis in Synchronized Cultures: II. Associations between the Nucleus and Chloroplasts at an Early Stage of the Cell Cycle under Photoautotrophic Conditions

Chloroplast-nucleus interactions were examined in cells of Euglenagracilis Z synchronized under photoautotrophic conditions. Thechloroplasts were localized near the cell periphery. At an earlystage of the cell cycle, however, some chloroplasts were transientlylocated in the inner space close to the nucleus. Electron microscopyusing serial cell sections revealed that the chloroplast formedprotrusions at several sites, which became associated with thenucleus. The outer membrane of the chloroplast envelope wasin contact, or at least continuous in part, with the outer membraneof the nuclear envelope at the sites of association, and densematerial was present in the chloroplast membrane. A chromosomewas close to each site of the association between these twoorganelles. Most of the chloroplasts including those in associationwith the nucleus were connected by fine bridges. The 4',6-diamidino-2-phenylindole-stainednucleoids in the chloroplast associated with the nucleus appearedto have a thread-like shape. There was another type of chloroplast-nucleusconnection, in which an intervening membranous body was in contactwith the outer part of the nuclear envelope on one side andwith the chloroplast envelope on the other side. ¹ This work was reported at the 48th Annual Meeting of the BotanicalSociety of Japan, Kyoto, October, 1983. (Received June 5, 1984; Accepted November 20, 1984)     

Interactions between the Nucleus and Cytoplasmic Organelles during the Cell Cycle of Euglena gracilis in Synchronized Cultures III. Association between the Nucleus and Chloroplasts at an Early Stage in the Cell Cycle under Photoorganotrophic Conditions (Part II)

Three-dimensional models of chloroplasts before and during theirassociation with the nucleus in Euglena gracilis Z were constructedbased on serial sections of cells taken during an early phaseof the cell cycle in synchronized cultures under photoorganotrophiccondition. Before association, the cell contained two to threechloroplasts which were composed of elongated tubular bodiesextending in different directions toward the cell periphery.These tubular arms of chloroplasts were drawn toward the nucleus,and folded, with concurrent coalescence, into a single giantbody surrounding the nucleus. A DAPI-fluorescence photomicrographof a giant chloroplast revealed that the chloroplast-nucleoidswere in the form of a continuous strand lying throughout thechloroplast body, and that some protuberances from the nucleoidstrand were in especially close proximity to the nuclear periphery. A new mode of the chloroplast-nucleus connection was observed.The nucleus had conspicuous protrusions, whose distal ends wereconnected with the chloroplast. The outer membrane of the nuclearenvelope was continuous with the outer chloroplast membrane,and at some sites, an open space was formed between the innernuclear membrane and the inner chloroplast membrane. ¹ This work was reported at the 48th Annual Meeting of the BotanicalSociety of Japan, held in Kyoto, in October, 1983. For PartI see Ehara et al. (1984).     

Interactions between the nucleus and cytoplasmic organelles during the cell cycle of Euglena gracilis in synchronized cultures. IV. An aggregate form of chloroplasts in association with the nucleus appearing prior to chloroplast division

Cells of Euglena gracilis were synchronized by applying a 14-h light:10-h dark regimen under photoautotrophic conditions and a 10-h light:14-h dark regimen under photoorganotrophic conditions. At a stage just prior to chloroplast division in the cell cycle of these synchronized cultures, chloroplasts temporarily gathered in the posterior part of the cell and were connected to each other by many bridges. Part of the chloroplast aggregate surrounded about half of the nuclear surface, making connections or close contacts at many sites. A chromosome was always attached to the inner membrane of the nuclear envelope at the site of association with the chloroplast. The nucleoids in these aggregate chloroplasts, examined by staining with 4',6-diamidino-2-phenylindole, a DNA fluorochrome, showed profiles of strings or strands with branchings, under photoorganotrophic conditions at least, and some parts of the branchings came close to the site of association with the nucleus. The association between the chloroplast aggregate and the nucleus was also observed in Euglena cells placed in continuous darkness after synchronization under photoorganotrophic conditions, suggesting that these organellar associations are related to the Euglena cell cycle but are not the result of light:dark alternations used for cell synchronization.     

Separation and Properties of the Inner and Outer Membranes of a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

The inner and outer membranes from a marine psychrophilic bacterium,Vibrio sp. ABE-1, were separated and purified by desalting,lysozyme treatment, and ultracentrifugation. Enzyme assay, electrophoresis,and chemical analysis showed that they had been highly purified.The isolated outer membrane had no 3-deoxy-D-mannooctulosonate(KDO) and chemical analysis revealed that it contained aboutthree times as much total lipid as the inner membrane. Consequently,the density of the inner membrane was higher than that of theouter membrane (1.19–1.23 gµcm³ for IM; 1.16–1.20g–cm³ for OM). The absolute majority of the lipids inthese membranes were phospholipids, more than half of whichwas identified as phosphatidylethanolamine. Virtually all cardiolipinwas found exclusively in the inner membrane, whereas an unidentifiedphospholipid containing hexosamine was only in the outer membrane.The ratios of unsaturated to total fatty acids in the innerand outer membranes were 76.0% and 69.6%, respectively. (Received February 23, 1984; Accepted August 8, 1984)     

Ontogenesis of Tannin Coenocytes in Sambucus racemosa L. I. Development of the Coenocytes from Mononucleate Tannin Cells

Tannin coenocytes in shoots of Sambucus racemosa L. developfrom mono-nucleate tannin cells which can be distinguished amongthe cells of the first internode, and which keep on growing.After karyokinesis without cytokinesis bi-nucleate tannin cellsoccur which yield a synchronous karyokinesis leading to 4 nucleiin the tube. The number of nuclei in the coenocyte is 2ⁿ wheren equals the number of karyokineses that have occurred. Sometimesthe number of nuclei is different and one nucleus is bigeerthan the rest indicating that a previous fusion of nuclei hasoccurred. The distribution of nuclei in the coenocyte supportsthe possibility of fusion of chromosome sets at the moment ofnuclear envelope dispersion. Sambucus racemosa L., coenocytes, synchronous karyokinesis, development     

Photo-induced H+ Transport between Chloroplasts and the Cytoplasm in a Protoplasmic Droplet of Characeae

Svintitskikh, V. A., Andrianov, V. K. and Bulychev, A. A. 1985.Photo-induced H⁺ transport between chloroplasts and the cytoplasmin a protoplasmic droplet of Characeae.—J. exp. Bot. 36:1414–1429. The effects of light on the membrane potential and cytoplasmicpH of isolated droplets of protoplasm from Nitella have beenstudied using microcapillary electrodes and pH-sensitive antimonymicro-electrodes. Illumination of chloroplast-containing dropletscaused a change of the membrane potential with a concomitantacidification of both the cytoplasm and the outer medium, butit had no effect on the electrical resistance of the surfacemembrane. Treatment of protoplasmic droplets with uncouplers(NH₄Cl and CCCP) resulted in a complete inhibition of the light-inducedacidification of the cytoplasm, whereas the energy transferinhibitor DCCD had no effect. A correlation between the formationof a pH gradient across the thylakoid membrane and the acidificationof the cytoplasm was explicable in terms of the assumption ofrestricted spatial communication between the intra-thylakoidvolume and the cytoplasm in intact chloroplast. The photo-inducedacidification of the boundary layer of an external medium wasmarkedly stimulated under the action of inhibitors of H⁺-ATPaseDCCD and DES. These findings suggest that the active extrusionof H⁺ from the cytoplasm into the external medium is not drivenby an ATPase, although H+-conducting channels of membrane ATPaseprovide a pathway for a passive diffusion of protons from outsideinto the cytoplasm Key words: Transport of protons, protoplasmic droplet, intact chloroplasts, Characeae     

Intracellular Photoreceptive Site for Blue Light-Induced Cell Division in Protonemata of the Fern Adiantum--Further Analyses by Polarized Light Irradiation and Cell Centrifugation

The intracellular localization of the photoreceptive site forblue light-induced cell division in single-celled protonemataof Adiantum capillus-veneris L. was investigated using polarizedlight irradiation and protonemal cell centrifugation. The responseto irradiation with polarized blue light showed no dependenceon the direction of light polarization. However, centrifugationof the protonemata followed by microbeam irradiation showedthat the site of blue light perception could be displaced togetherwith the nucleus. Centrifugal treatment changed the distributionof intracellular organelles at the time of light exposure andbasipetally displaced the nucleus about 90µm. This treatmenthad no effect on the induction of cell division with blue lightif the protonemata were centrifuged again acropetally afterthe light treatment. Microbeam (30x30 µm²) irradiationwith blue light of the apical 45–75 ßm region,the receptive site of blue light in non-centrifuged cell, didnot induce cell division. However, cell division was inducedby irradiation of the nucleus-containing region, indicatingthat the photoreceptive site was displaced together with thenucleus by the centrifugation. These results suggest that theblue light receptor regulating cell division in Adiantum protonematais not likely to be located on the plasma membrane. (Received February 20, 1986; Accepted May 27, 1986)     

An Alternative Electron Transfer Pathway Mediated by Chloroplast Envelope

Localization of redox active substance(s) in chloroplast envelopeswas revealed by means of the oxidation of Cyt c by isolatedouter and inner envelope preparations. Irradiated chloroplastsreduced extra-chloroplastic Cyt c probably by an envelope electrontransfer chain. The rate was saturated at a level of about 10µmol (mg Chl)^–1 h^–1 under weak light of 10µEm^–2 s^–1. Cyt c photoreduction was inhibited by DCMUbut not by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)indicating that the plastoquinone site is the junction of photosyntheticelectron transfer chain to envelope redox substance. Completesuppression of the non-cyclic photophosphorylation of thylakoidsby the presence of envelope membranes indicates that there isan alternative electron transfer path-way in envelope membranesthat bypasses over the pH-forming plastoquinone shuttle in thephotosynthetic electron transfer chain. ¹ Present address: Photosynthesis Research Laboratory, Instituteof Physical and Chemical Research (RIKEN), Wako, Saitama, 351-0198Japan.     

Apoptotic Cell Death and Cellular Surface Negative Charge Increase in Maize Roots Exposed to Cytotoxic Stresses

Maize root meristematic tissues were exposed to cytotoxic reagents,the RNA-synthesis inhibitor Actinomycin D (ActD), the protein-synthesisinhibitor cycloheximide (CHX) and the mitosis inhibitor colchicine(COL). Morphological and biochemical evidence of specific apoptoticnuclei and chromosomes in individual treated cells was identifiedusing a simple and highly efficient chromosome spreading-basedTUNEL assay, DNA laddering and DNA gel blotting. All of thesedrugs induced DNA cleavage, dose-dependent oligomeric ladders,and characteristic nuclear and chromosomal condensations. Resultsfrom DNA gel blotting showed that DNA ladders could be inducedby exposure to 0.1 mg l^-1ActD, 100 mg l^-1CHX and 500 mg l^-1COLfor 6 h, 6 h and 12 h respectively. The sequence of changesin single cells was studied in detail. DNA cleavage was foundto occur before condensation and disorganization of the nucleus,followed by deformation and condensation of metaphase chromosomes,and marginalization of chromatin. Finally, nucleoli disappearedand fragmentation of the nucleus occurred. Meanwhile, changesin the outer surface charge of apoptotic cells were assessedby electrophoresis. Results indicated quantitatively that thesurface negative charge increased during these apoptotic processes.Our results also showed that the apoptotic pathway induced byeach of these drugs could be reversed before serious cleavageof DNA into oligonucleosomal fragments and universal chromatincondensation. Copyright 2001 Annals of Botany Company Cytotoxin, chromosome spreading, apoptosis, cell electrophoresis     

NUCLEAR ENVELOPE-CHLOROPLAST RELATIONSHIPS IN ALGAE

In Ochromonas danica and two related species (Chrysophyceae) and in Rhodomonas lens and Cryptomonas sp. (Cryptophyceae), the chloroplast is surrounded by an outer double-membraned envelope which lies outside the usual double-membraned chloroplast envelope. At the borders of the area where the chloroplast lies adjacent to the nucleus, this outer envelope is continuous with the outer membrane of the nuclear envelope as a double-membraned outfolding, so that the entire chloroplast in these species lies within a double-membraned sac, one wall of which is the nuclear envelope. In Olisthodiscus sp. (Chrysophyceae ?), each of the small peripheral chloroplasts is surrounded by a similar double-membraned outer envelope, but in this species no connections with the nuclear envelope were observed. In the Ochromonadaceae, a characteristic array of tubules is present within the sac in the narrow space which separates the chloroplast from the nucleus. In the other species studied, tubules are present at places between the chloroplast envelope and the outer envelope. In the Cryptophyceae, the starch grains lie outside the chloroplast envelope, but within the outer double-membraned sac. A double-membraned outer envelope appears to be present outside the chloroplasts of the Phaeophyta and Euglenophyta, but seems to be absent in the other groups of algae.     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

Cell Cycle Timing of Replication of the Nuclear Gene Encoding Chloroplastic NADP-Specific Glutamate Dehydrogenase Isoenzymes in Chlorella sorokiniana

In synchronized Chlorella sorokiniana cells, the NH₄⁺ inducibleNADP-specific glutamate dehydrogenase enzyme (NADP-GDH) accumulatedin a linear manner throughout the first cell cycle. Early inthe following second cell cycle, an increase in its rate ofaccumulation occurred that was proportional to the increasein total cellular DNA in the previous cell cycle. In synchronizedbacterial cells, increases in rate of linear accumulation ofinducible enzymes coincide with the time of replication of theirstructural genes. To determine whether the rate change in NADPGDHaccumulation resulted from a delay in replication of its nuclearstructural gene (gdhN) in fully induced C. sorokiniana cells,the cell cycle timing of replication of this gene was comparedto that of another nuclear gene, nitrate reductase (nia), andof a chloroplast gene, ribulose bisphosphate carboxylase large-subunit(rbcL), in synchronized cells cultured in NH₄⁺ or NO₃^–(uninduced) medium. The gdhN and nia genes replicated withinthe period of nDNA synthesis and rbcL within the period of ctDNAsynthesis in cells growing in either nitrogen source. Therefore,the delayed rate change in enzyme accumulation results froma process that regulates expression of the gdhN gene after itsreplication. (Received July 16, 1994; Accepted November 28, 1994)     

Destruction of Chloroplast Nuclei of the Male Gamete by Calcium and Nuclease C In a Cell Model of Chlamydomonas reinhardtii

A cell model was prepared for each mating type cell of Chlamydomonasreinhardtii in order to investigate the molecular mechanismfor the maternal inheritance of chloroplast genes. Chloroplastnuclei (cp-nuclei) in the chloroplast were well preserved. Therefore,their disappearance after a change in the external medium couldbe monitored by high resolution fluorescence microscopy afterstaining the cells with 4'-6-diamidino-2-phenyl indole. Thenumber of cp-nuclei in the cell model diminished when Ca²⁺ wasadded to a cell suspension. Presumably, this ion activated theendogenous nuclease C that remained in the cytoplasm, thus settingup the destruction of chloroplast DNA. Almost all disappearedwhen Ca²⁺ was given together with nuclease C. From the kineticsof the disappearance of cp-nuclei after combining the cell modelwith nuclease C, we found that male gametes begin to lose theircp-nuclei after 20 min, whereas female gametes lose them after40 min. Furthermore, as a culture aged, the female cp-nucleibecame resistant to the destructive attack by nuclease C, butthe male cp-nuclei remained sensitive. (Received October 8, 1984; Accepted January 21, 1985)     

The chloroplast envelope of Phaseolus vulgaris L. II. Enzymic characteristics

The isolated chloroplast envelope of Phaseolus vulgaris L. wasfound to synthesize galactolipids when supplied with exogenousUDP galactose. The rate of total galactose incorporation variedwithin the range 0.4-50 nmole g protein^–1 sec^–1with age of the tissues. The activities of NADH dehydrogenase,malate dehydrogenase and Mg²⁺-ATPase in the envelope fractionwere also investigated. The use of a positive "marker" activityfor the isolated chloroplast envelope is considered. ¹Present address: Boyce Thompson Institute, Tower Road, CornellUniversity, Ithaca, NY 14853, USA. (Received March 24, 1980; )     

Polypeptide Compositions of Stroma, Internal Membranes, and Inner and Outer Envelope Membranes of Amyloplast from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Intact amyloplasts isolated from liquid-cultured white-wildcells of sycamore (Acer pseudoplatanus L.) were further subfractionatedinto internal membranes (d=1.05g/ml), envelope membranes (d=1.12g/ml)and stromal fraction, which contained each characteristic polypeptidecomposition as revealed by the Na-dodecyl sulfate polyacrylamidegel electrophoresis. Absorption spectra of internal and envelopemembranes were distinctly different. By the immunoblotting analysis,it was shown that the amyloplast envelope membranes contain31 kDa P_i-translocator, although it is not the predominant polypeptidecomponent in contrast to the case of chloroplast envelope. Onehundred kDa -l,4-glucan phosphorylase (plastid type) was detectedin the stromal fraction of amyloplasts using the specific antibodyraised against the major form of -l,4-glucan phosphorylase frompotato tuber. Amyloplast envelopes were further separated into inner and outermembrane fractions by the freezing-thawing method originallydeveloped for the separation of chloroplast envelope membranesby Cline and associates (1981) (Proc. Natl. Acad. Sci. USA 78:3595–3599). Nadodecylsulfate gel electrophoretic analysisrevealed that the inner and outer envelope membranes containthe distinctly different polypeptide compositions. ¹Supported by grants from the Ministry of Education, Scienceand Culture (Mombusho) of Japan. This is paper No. 77 in theSeries "Structure and Function of Chloroplast Proteins". ²Recipient of a predoctoral student fellowship from the Japanesegovernment (Mombusho). Permanent address: Department of Biochemistry,Faculty of Science, Kasetsart University, Bangkok 10900, Thailand ³Permanent address: Department of Biology, Southwest AgriculturalUniversity, BeiBei Chongqing, People's Republic of China. Holderof the Chinese Government Scholarship (1987) (Received May 27, 1988; Accepted August 30, 1988)     

Turnover of Cell Wall Polysaccharides during the Cell Cycle in a Synchronous Culture of Catharanthus roseus

Pulse-chase experiments were done using a synchronous cultureof Catharanthus roseus in order to study cell wall turnoverduring the cell cycle. [¹⁴C]Glucose was fed for 1 h to cells35 and 49 h after the re-start of the cell cycle. Radioactivitywas then diluted with a large amount of cold glucose and chasedduring the early G1 phase after the first cell division, thetime at which an increase in the amount of cell walls mainlytook place. A pulse-chase with [¹⁴C]glucose was also made duringthe S phase when cell walls had not increased so much. Radioactivity of the EDTA-soluble (pectin) fraction decreasedduring the chase in the early G1 phase; whereas, the radioactivitiesof the other cell wall fractions, as well as extracellular polysaccharide(ECP) increased during the chase, both in the early G1 and inthe S phases. The radioactivity of uronic acid in ECP was higherin the early G1 phase than in the S phase. These results indicatethat an active turnover of pectin may take place in the earlyG1 phase after the first cell division. ¹ Present address and reprint requests: Biological Institute,Tohoku University, Sendai 980, Japan. (Received November 5, 1984; Accepted April 2, 1985)     

Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes

Outer and inner envelope membranes of spinach chloroplasts wereisolated using floatation centrifugation followed by sedimentationsucrose density gradient centrifugation after disruption ofintact chloroplasts by freezing and thawing. Two major fractionswith buoyant densities of 1.11 and 1.08 g cm^–3 and a minorfraction with a density of 1.15 g cm^–3 were obtained.They were identified as innei and outer envelope and thylakoidfractions, respectively, by analyzing their polypeptide compositionby high-resolution SDS-PAGE and the N-terminal sequences oftheir protein components. Due to the refinement of the isolation procedure, most of theribulose-l,5-bisphosphate carboxylase/oxygenasc (RuBisCO), whichhad always been observed as a contaminant, was eliminated fromthe outer envelope fraction. Application of high-resolutionSDS-PAGE revealed that this fraction was rich in the low-molecular-massouter envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl.Acad. Sci. USA 87: 5778] and a protein with a molecular massof 15 kDa which is homologous to the 16 kDa outer envelope proteinof pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94:9504]. The two proteins account for 90% of the total proteinspresent in outer envelope membranes. Proteins which are suggestedto function in translocation of nuclear-encoded polypeptideswere not identified in the envelopes from spinach in the presentstudy. Differences in the protein composition of outer envelopemembranes arc discussed based on the developemental stages ofchloroplasts. ¹Present address: Biological Function Section, Kansai AdvancedResearch Center, Communications Research Laboratory, Ministryof Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan.     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)