Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. II. Disassembly and Reassembly of Proplastid-Nuclei Isolated from Cultured Cells

The effects of various concentrations of NaCl on the compactstructure of proplastid-nuclei (pp-nuclei) isolated from culturedtobacco cells were examined by fluorescence microscopy usinga DNA-specific fluorochrome, 4, 6-diamidino-2-phenylindole.Simultaneously, behavior of the four proplastid DNA-bindingproteins (mol wt: 69kDa, 31 kDa, 30kDa and 14kDa) identifiedpreviously (Nemoto et al. 1988) was investigated by SDS-polyacrylamidegel electrophoresis. When the concentration of NaCl was increased, the isolated pp-nucleiwere gradually dispersed, and at concentrations greater than0.4 M NaCl they were almost completely dispersed. During thisdisassembly process, the 31-kDa and 30-kDa proteins dissociatedfrom the pelletable pp-nuclear fraction to the supernatant at0.1 M NaCl, whereas the 69-kDa and 14-kDa proteins dissociatedto the supernatant only at 0.4 M NaCl. When the concentrationof NaCl was decreased again by dialysis, the pp-nuclei whichhad been dispersed by 2 M NaCl were gradually reassembled intocompact structures which were almost identical to the originalpp-nuclei. During this reassembly process, the 69-kDa and 14-kDaproteins first returned to the pellet fraction, and subsequentlythe 31-kDa and 30-kDa proteins moved into the pellet. This behavior of the proplastid DNA-binding proteins stronglysuggests that the association of these proteins with plastid-DNAis responsible for the compact organization of pp-nuclei. Inaddition, it was indicated that the 69-kDa and 14-kDa proteinsare more tightly bound to plastid-DNA than are the 31-kDa and30-kDa proteins. ⁴Present address: Biological Laboratory, Faculty of Science,Nara Women's University, Nara, 630 Japan. (Received August 24, 1988; Accepted February 28, 1989)     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. III. Isolation of Chloroplast-Nuclei from Mesophyll Protoplasts and Identification of Chloroplast DNA-Binding Proteins

We have developed a method of isolating morphologically intactchloroplast-nuclei (nucleoids) in large quantities from mesophyllprotoplasts of Nicotiana tabacum L. cv. Bright Yellow-2. The isolated chloroplast-nuclei (cp-nuclei) were dispersed bythe treatments with proteinase K, 2 M NaCl and 2 M KCL, whichsuggested that the cp-nuclei are compactly organized by an electrostaticinteraction between the chloroplast-DNA and some protein(s).However, the four proplastid DNA-binding proteins identifiedpreviously (Nemoto et al. 1988) were not found in the cp-nuclei,and a different set of DNA-binding proteins (mol wt: 35 kDa,28 kDa and 26 kDa) was detected in the cp-nuclei by a DNA-bindingassay. On the other hand, the chloroplast-DNA was not differentfrom the proplastid-DNA. These findings indicate that the cp-nuclei are constituted fromthe plastid-DNA and the chloroplast-specific DNA-binding proteins.This suggests that the DNA-binding proteins in proplastids aredynamically replaced with the chloroplast DNA-binding proteinsduring the differentiation of plastids from proplastids to chloroplasts.The change of DNA-binding proteins may be involved in the morphologicalchange of plastid-nuclei and/or the regulation of plastid-DNAreplication and gene expression during the differentiation processof plastids. ⁶Present address: Department of Biotechnology, Faculty of Technology,Tokyo University of Agriculture and Technology, Nakamachi, Koganei,Tokyo, 184 Japan ⁷Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Tokyo, 113 Japan (Received April 4, 1990; Accepted May 18, 1990)     

Changes in Levels of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum L.

Expanded leaves of Mesembryanthemum crystallinum L. performingC₃ photosynthesis were induced to perform pronounced Crassulaceanacid metabolism (CAM) by exposing the plant roots to higherNaCl concentration. Levels of phosphoenolpyruvate (PEP) carboxylaseactivity increased 10-fold during the 7-day induction period.Densitometric analysis of Coomassie-stained sodium dodecyl sulfate(SDS) polyacrylamide gradient slab gels of leaf extracts, preparedduring the course of CAM induction, revealed that at least fivebands of polypeptides increased in content (kilodalton valuesof 98, 91, 45, 41, 38). Higher levels of three additional polypeptides(kilodalton values of 102, 76, 33) became apparent after tissuehad been grown for 2 weeks at 400 mM NaCl. Of these polypeptides,that having a mass of 98 kilodaltons was identified as the subunitof PEP carboxylase by comparison with the corresponding bandfrom partially purified PEP carboxylase from the same tissue.Only a faint 98 kilodalton band was evident on SDS gels fortissue operating in the C₃ mode; staining intensity at thislocation increased with increasing NaCl-salinity in the rootingmedium until CAM was fully induced. These data provide evidencefor net synthesis of PEP carboxylase and several other proteinsduring the induction of CAM in M. crystallinum. ¹ Present address: USDA, P. O. Box 867 Airport Rd., Beckley,WV. 25801, U.S.A. ² Present address: Department of Botany, Washington State University,Pullman, Washington 99164, U.S.A. ³ Present address: Botanisches Institut der Universit?t, MittlererDallenbergweg 64, 8700 W?rzburg, W.-Germany. (Received October 27, 1981; Accepted March 15, 1982)     

Effect of low concentration of hydrogen peroxide on indoleacetate oxidase zymogram in Pharbitis nil

Indoleacetate oxidase activity in Pharbitis nil, Japanese morningglory, was zymographically examined. Some isozymes appearedonly after treatment with a low concentration of hydrogen peroxide.This phenomenon was assumed to be due to the overlapping ofboth isozymes and natural inhibitor substances on the zymograms. ¹Present address: Biological Institute, Department of LiberalArts, Shizuoka University, Ooya 836, Shizuoka, Japan. (Received September 30, 1968; )     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. IV. Association of Chloroplast-DNA with Proteins at Several Specific Sites in Isolated Chloroplast-Nuclei

When chloroplast-nuclei (cp-nuclei), isolated from tobacco mesophyllprotoplasts, were directly digested by restriction enzymes andanalyzed by agarose gel electrophoresis, the migration of severalrestriction fragments was greatly inhibited during the electrophoresis.When the digested cp-nuclei were treated with SDS and proteinaseK prior to electrophoresis, all the restriction fragments migratednormally and the electrophoretic pattern was the same as thatgenerated by similarly restricted, purified chloroplast-DNA(cp-DNA). Use of a newly developed method, "reciprocal electrophoresis",proved that these fragments were bound so tightly to certainDNA-binding protein(s) that the fragments were trapped in thegel slot. These fragments corresponded to at least four sitesof the cp-DNA. By contrast, when proplastid-nuclei (pp-nuclei)isolated from tobacco cultured cells were analyzed in the sameway, no restriction fragments were trapped in the gel slot.These results, taken together, suggest that a DNA-binding protein(s)is present in cp-nuclei, but not in pp-nuclei, and binds tightlyto the four sites of the cp-DNA. Therefore, the molecular architectureof cp-nuclei is clearly different from that of pp-nuclei. TheDNA-binding protein(s) may have some function(s) specific tocp-nuclei. ⁴ Present address: Department of Biotechnology, Faculty of Technology,Tokyo University of Agriculture and Technology, Nakamachi, Koganei,Tokyo, 184 Japan (Received July 30, 1990; Accepted November 29, 1990)     

EUGLENA GRACILIS IN SYNCHRONOUS DIVISION I. DRY MASS AND VOLUME CHARACTERISTICS

Volume distributions and dry mass have been measured in synchronouspopulations of Euglena gracilis, grown in a salt medium at aconstant temperature. In keeping with the approximate doublingof cell number observed in each division burst, the averagedry mass and volume are doubled in each inter-division period. ¹ Present address: Department of Biology, Tokyo MetropolitanUniversity, Setagaya.ku, Tokyo. (Received February 17, 1961; )     

Studies on chlorophyll formation in Chlorella protothecoides III. Effects of chloramphenicol, cycloheximide, and ethionine on chlorophyll formation

Effects of chloramphenicol, cycloheximide, puromycin and ethionineon the light-independent and subsequent light-dependent processesof chlorophyll formation in "glucose-bleached" cells of Chlorellaprotothecoides were studied. These substances, except puromycin,strongly suppressed different phases of chlorophyll formation.Ethionine most strongly suppressed the light-independent phaseand chloramphenicol an early, relatively short process in thelight-dependent phase of chlorophyll formation. Cycloheximideseverely suppressed all phases of chlorophyll formation. Possibleimplications of these results for the biosynthesis of chlorophyllin algal cells are discussed. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. ² Laboratory of Entomology, Faculty of Agriculture, TamagawaUniversity, Machida-shi, Tokyo, Japan (Received October 5, 1972; )     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)     

Electrical Characteristics of the Tonoplast of Cham corallincr. A Study Using Permeabilised Cells

Use of permeabilised cells of Chara corallina provides a uniqueopportunity to study the electrical characteristics of the tonoplastwhilst being able to control ionic conditions on the outsideof the membrane. Current-voltage (I/V) analysis over wide voltagespans, and admittance measurements at 5 Hz showed that manypermeabilised cells had a similar conductance and capacitanceto the tonoplast of intact cells. Cells developed two regionsof negative-slope conductance upon addition of external Cl^–,which suggests the existence of potential-dependent Cl^–channels in the Chara tonoplast. With Cl^– concentrationssimilar to those expected in vivo, the resting potential wasmore sensitive to changes in external K⁺ than Cl^–; however,a decrease in external K⁺ did not significantly alter the shapeof the I/V relation. ¹Present address: Biopysics Laboratory, School of BiologicalSciences, A12, University of Sydney, Sydney, N.S.W., 2006, Australia ²Permanent address: Department of botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan (Received May 6, 1987; Accepted September 21, 1987)     

Induction of Cold Hardiness by Salt Stress Involves Synthesis of Cold- and Abscisic Acid-Responsive Proteins in Potato (Solanum commersonii Dun)

Salt stress has been found to increase frost tolerance in someherbaceous species. In an attempt to understand the molecularbasis of the frost tolerance induced by salt stress, the effectof salt (100 mM NaCl) on total proteins in stem-cultured potatoplantlets (Solarium commersonn Dun) was analyzed on two-dimensionalgels. Nine salt-induced proteins were identified after 24 hsalt treatment, at which time cold hardiness increased by threedegrees. Direct comparisons of the proteins with those inducedby cold- and abscisic acid(ABA)-treatments revealed that fiveof the salt-induced proteins were also induced by cold(4°/2°C)-treatmentand seven were also induced by ABA(40µM)-treatment. Threeproteins (M_rr/pls 13/7.0, 27/6.6 and 48/6.9) were induciblein both cold- and ABA-treatments in association with frost hardening.After 6 h salt treatment, endogenous ABA levels in plantletleaves showed a transient six-fold increase before cold hardinessdeveloped. The results suggest that salt-induction of cold hardinessinvolves the synthesis of cold and ABA-responsive proteins andthe alteration of protein synthesis is mediated by ABA elevatedupon salt stress. This study also suggests that a subset ofproteins induced by cold and ABA-treatments are related to saltstress. ¹Scientific Journal Series Paper No. 20787 of the MinnesotaAgricultural Experimental Station, St. Paul, MN 55108, U.S.A. ²Present address: Department of Biochemistry Willard Hall, KansasState University, Manhattan, KS 66506, U.S.A. ³Present address: Via G.B. Marino 13, 80125-Napoli, Italy ⁴Present address: Institute of Biological Chemistry, WashingtonState Univ., Pullman, WA 99164, U.S.A.     

COMPARATIVE STUDIES ON GROWTH, RESPIRATION, PHOTOSYNTHESIS AND PIGMENT CONTENT IN RHODOPSEUDOMONAS PALUSTRIS

1. Comparative studies were performed on growth, photosyntheticand respiratory activities, and pigment content in Rhodopseudomonaspalustris.
2. The growth of the organism, as influenced by variousculturalconditions such as light, aerobiosis, anaerobiosisand nutritionalfactors was investigated.
3. The respiratoryactivity of the bacterium was found to be higherin dark-growncells than in cells grown in the light. The photosyntheticactivitydid not significantly depend on the growth conditionsof theculture. Cells of younger cultures were found to be moreactivethan those of older cultures, with respect both to respirationand photosynthesis.
4. The pigment content was found to be higherin the light-growncells than in the dark-grown ones. The ratiophotosyntheticactivity/bacteriochlorophyll was significantlyhigher in thelatter than in the former.
5. Light, as well asvarious nutritional factors, was found toexert a marked accelerationon pigment formation, although ithas not yet been possibleto culture cells completely lackingin photosynthetic pigmentsand accordingly in photosyntheticactivity.

¹ Present address: Division of Dermatology and Urology, TokyoMetropolitan Hiroo Hospital, Tokyo. ² Present address: Department of Biology, Saitama University,Urawa. ³ Present address: Department of Biochemistry, School of Medicine,Yokohama University, Yokohama. ⁴ Present address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Tokyo. (Received July 23, 1961; )     

Analysis of Lipids in Prochloron sp.: Occurrence of Monoglucosyl Diacylglycerol

Prochloron contained monogalactosyl diacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyl diacylglycerol, phosphatidylglyceroland, as a minor component, monoglucosyl diacylglycerol, butno phosphatidylcholine. With respect to the lipid and fattyacid compositions, this alga is similar to the blue-green algaerather than the chloroplasts of eukaryotic plants. ¹ Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received August 24, 1982; Accepted November 18, 1982)     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

Plant Cell Wall Proteins: Partial Characterization of Maize Wall Proteins With Putative Roles in Auxin-Induced Growth

Antibodies raised against cell wall proteins inhibited auxin-inducedgrowth of Zea mays L. coleoptile segments. The total complementof proteins isolated from the cell walls of Zea mays seedlingswas fractionated by cation exchange and gel filtration chromatography.A procedure was developed to evaluate these cell wall-proteinfractions for their ability to reverse growth inhibition causeby specific antibody binding. Inhibition of growth was attributedto specific antibody-antigen interaction based on the observationsthat only serum containing antibodies against certain cell wallproteins inhibited growth, that gamma globulins purified fromappropriate serum samples inhibited growth, and that a specificsubfraction of isolated cell wall proteins precipitate the growthinhibiting antibody. Antigens which generated growth inhibitoryantibodies were identified as an acidic group of proteins withapparent relative molecular masses in the range of 20–25kDa. This subfraction of cell wall proteins was not effectivein hydrolyzing cell wall polysaccharides. A small amount ofcarbohydrate was found associated with this fraction and mayreflect some degree of glycosylation of some of the proteins ¹Supported in part by National Science Foundation Research GrantPCM 7818588 ²Present Address: USDA-ARS, U.S. Dairy Forage Research Center,University of Wisconsin, Madison, WI 53706 ³Present Address: Department of Vegetable Crops, Universityof California, Davis, CA 95616 (Received November 2, 1987; Accepted March 31, 1988)     

Effects of Leaf Chilling on Thylakoid Functions, Measured at Room Temperature, in Cucumis sativus L. and Oryza sativa L.

Photosynthetic functions in leaves of cucumber (Cucumis sativusL.) and rice (Oryza sativa L.) were examined before and aftervarious chilling treatments. Cucumber leaves lost the capacityfor the photosynthetic oxygen evolution after chilling at 0°Cin the dark for 48 h. Thyla-koids isolated from such leaveswere not able to reduce dichloroindophenol (DCIP), but the additionof diphenylcarbazide (DPC), an electron donor to PS II, restoredthe ability to reduce DCIP, indicating that the site of damageis in the water-splitting machinery of PS II. In moderate light (500 jumol quanta m^–2s^–1), chillingof cucumber leaves at 5°C for 5 h was sufficient to inducethe complete loss of the capacity for photosynthetic oxygenevolution. Electron transport rates measured in thylakoids wereunaltered, but thylakoids were totally permeable to protons.Since the addition of dicyclohexylcarbodiimide (DCCD) restoredcoupling and the capacity for proton uptake, the primary siteof damage was deduced to be in the ATPase. In rice, both chilling treatments had barely any effect on thylakoidfunctions, although some negative effects was apparent in photosynthesisin leaves. ¹Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. ²Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received January 11, 1989; Accepted June 12, 1989)     

SOME PROPERTIES OF THE CYTOCHROME C REDUCING SUBSTANCE, A FACTOR FOR LIGHT-INDUCED REDOX REACTION OF CYTOCHROME C IN PHOTOSYNTHETIC LAMELLAE

Cytochrome c reducing substance (CRS), a redox substance discoveredin photoreactive lamellar fragments, was purified by Sephadexcolumn chromatography. Chromatographic behaviours of CRS ofAnabaena and spinach were essentially the same. Purified CRSof Anabaena showed an absorption spectrum having one absorptionmaximum around 260 mµ. The absorption peak disappearedon addition of excess amount of borohydride. Similar absorptionchange on borohydride addition was observed with spinach CRSpreparation. Purified preparations of Anabaena and spinach CRS supportedphotophosphorylation in spinach broken chloroplasts. The phosphorylationwas found to couple the electron flow from water to molecularoxygen. ¹This work was supported by grant GM-11300 from the NationalInstitute of Health, U. S. A. ²Present address: Institute of Applied Microbiology, The Universityof Tokyo, Tokyo, Japan.     

Demonstration of light-induced potential change in Chara cells lacking tonoplast

Chara cells without tonoplasts, prepared by replacing the cellsap with EGTA-containing media, showed essentially the samepattern of light-induced changes in membrane potential and membraneresistance as normal cells although the concentrations of ionsand ATP in the cytoplasm decreased considerably (1/3–1/10)after loss of the tonoplast. Removal of the tonoplast reducedthe rate of photosynthetic O₂ evolution to about 50% of thatof normal cells but did not affect the magnitude of light-inducedpotential change. Not a full but a certain level of electronflow seems necessary to activate the putative electrogenic H⁺-pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Japan. ² Present address: Niigata College of Pharmacy, Niigata 950-21,Japan. (Received September 4, 1978; )     

Effects of Osmotic Stress, Ionic Stress and IAA on the Cell-Membrane Resistance of Vigna Hypocotyls

The cell-membrane resistance (R_m) of Vigna hypocotyls was examined,and the effects of osmotic stress, ionic stress and IAA on R_mwere investigated. R_m decreased by 64 to 77% under osmotic stressin the presence of absorbable solutes (40 mM sorbitol, 15 mMKC1, 30 mM sucrose; or 40 mM sorbitol, 15 mM KC1, 30 mM sucroseplus 10^–4 M IAA) or under ionic stress (50 mM NaCl or50 mM KC1). R_m was not changed by perfusion with 10^–4M IAA. Therefore, the hyper-polarizations of the membrane potentialobserved in both cases should be ascribed totally to the activationof the electrogenic proton pump. Although R_m showed an increaseof 1.6 fold when the hypocotyls were subjected to osmotic stress(100 mM sorbitol or 100 mM sorbitol plus 10^–4 M IAA),83.6% or 92.4% of the hyperpolarization of the membrane potential(V_px was also the result of the activation of the pump. Theresults, calculated on the basis of the current source model,support the viewpoint that the hyperpolarization of the cellmembrane potential of Vigna hypocotyls under osmotic stress,ionic stress or in the presence of IAA is an expression of theactivation of the proton pump, and is not caused by an increasein R_m. ¹ Present address: Researchers and Planners of Natural Environment, Yotsugi Bldg. (2F), 1-5-4 Horinouchi, Suginami-Ku, Tokyo,166 Japan ² Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2 Seto, Kanazawa-Ku, Yokohama, 236 Japan (Received February 14, 1991; Accepted July 24, 1991)     

Nucleotide Sequence of cDNA Clones Encoding the PS I-H Subunit of Photosystem I in Tobacco

cDNA clones encoding the PS I-H subunit of photosystem I wereisolated from Nicotiana tabacum and Nicotiana sylvestris. Thenucleotide sequences of three clones showed that, in both species,the mature PS I-H protein consists of 95 amino acid residuesand has a calculated molecular mass of 10.3 kDa. ³ Present address: The Institute of Physical and Chemical Research,Tsukuba, 305 Japan.