Properties of the Respiratory NAD(P)H Dehydrogenase Isolated from the Cyanobacterium Synechocystis PCC6803

Activity staining with NADPH-nitroblue tetrazolium after native-PAGEof membrane proteins of Synechocystis PCC6803, solubilized with3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),revealed four NAD(P)H dehydrogenase (NDH) activities; an NDHcomplex of the respiratory chain, a ferredoxin NADP⁺ reductase(FNR), a drgA product which oxidized both NADH and NADPH, andan uncharacterized NADH-specific enzyme. The NDH complex waspurified with anion exchange and gel filtration chromatographies.The purified complex had a molecular mass of 376 kDa and wascomposed of 9 subunits. Western analysis showed that the complexcontained the NDH-H subunit, but not NDH-A or B. The enzymereduced ferricyanide much faster than plastoquinone and usedNADPH as its prefered electron donor rather than NADH. The enzymaticactivity was inhibited by diphenyleneiodonium chloride and salicylhydroxamicacid, but not by rotenone, p-chloromercuribenzoate, N-ethylmaleimide,flavon, dicumarol, or antimycin A. These results suggest thatthe purified complex is a hydrophilic subcomplex which containsan NADPH binding site and flavin, and is dissociated from ahydrophobic subcomplex, which contains quinone binding site. ¹Present address: Division of Applied Life Sciences, GraduateSchool of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502Japan ³Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuencho, Fukuyama, Hiroshima, 729-0292Japan     

Metabolism of p-Hydroxybenzoate via Hydroxyquinol by Trichosporon cutaneum WY2-2: Characterization of the Pathway using Superoxide Dismutase as a Stabilizer of Hydroxyquinol

Trichosporon cutaneum WY2-2 was shown to metabolize p-hydroxybenzoatevia protocatechuate and hydroxyquinol. Using superoxide dismutaseas a stabilizer of hydroxyquinol, the conversion of protocatechuateto hydroxyquinol and the ring fission process of hydroxyquinolwere confirmed. Hydroxyquinol was chemically identified as theproduct of protocatechuate hydroxylase reaction. Partially purifiedprotocatechuate hydroxylase was highly specific for protocatechuate;its K_m values for protocatechuate and NADH were 17.6 and 12.4µM, respectively. It catalyzed equimolar CO₂ formation,NADH oxidation and O₂ consumption from protocatechuate. Hydroxyquinoldioxygenase was highly specific for hydroxyquinol, with a K_mof 2.9 µM. ¹A preliminary account of this work was presented at the 81stMeeting of the Chubu-branch of Agricultural Chemical Societyof Japan, Gifu, October, 1980. ²Present address: Biological Institute, Faculty of Science,Nagoya University, Nagoya 464, Japan. ³Present address: Shin Nihon Chemical Co. Ltd... 19-10, Showa-cho,Anjoh, Aichi 446, Japan. (Received November 15, 1985; Accepted August 27, 1986)     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

Relationship between light-induced potential change and internal ATP concentration in tonoplast-free Chara cells

The effect of the intracellular concentration of ATP ([ATP]₁)on the light-induced potential change (LPC) in tonoplast-freeChara cells was studied. The LPC was hardly affected by loweringthe [ATP]₁ by about 1/10 or by raising it to about 10 timesthe normal cytoplasmic concentration (0.5–1.3 mM). Theinsensitivity of LPC to [ATP]₁ excludes the possibility thatan increase in [ATP]₁ due to photosynthesis may induce the LPC.However, extreme lowering of the [ATP]₁ to about 1–2 µMcompletely inhibited LPC, although photosynthetic O₂ evolutionwas not significantly inhibited. This fact supports the hypothesisthat light stimulates the putative H⁺pump fueled by ATP. Theuncoupling agents DNP and CCCP greatly depolarized the membrane,and inhibited LPC strongly, but they did not decrease [ATP]₁.Photosynthetic O₂ evolution was inhibited to some extent by2 µM CCCP and strongly inhibited by 0.1 mM DNP. Sincethe membrane resistance increased significantly, these chemicalsare believed to act on the membrane as an inhibitor of the electrogenicH⁺ pump not as an H⁺conductor. Introduction of 1 mM ATP intocells treated with uncouplers, to a large extent restored theirability to produce LPC although the membrane potential in darknesswas maintained at a low level. ¹Present address: Niigata College of Pharmacy, 5829 Kamishinei-cho,Niigata 950-21, Japan. ²Present address: Department of Agricultural Chemistry, Collegeof Agriculture, Kyoto University, Kyoto 606, Japan. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received March 9, 1979; )     

NO2 Suppression of Light-Induced Nitrate Reductase in Squash Cotyledons

NADH-nitrate reductase (NR) (EC 1.6.6.1 [EC] ) activity in the cotyledonsof squash (Cucurbita maxima Duch.) seedlings showed daily variationwhen the seedlings were subjected to an alternating light-darkcycle. When the seedlings were transferred into continuous darkness,NR activity rose at first and then decreased continuously. Irradiationafter continuous darkness induced a rapid increase in NR activity;this light induction of NR activity was inhibited completelyby fumigation with 4 ppm nitrogen dioxide (NO₂). This inhibitoryeffect of NO₂ was prominent even at 1 ppm and became more pronouncedas the concentration of NO₂ increased. NO₂ fumigation did notremarkably affect the content of reductant (NADH) in the cotyledons.The results of immunoblotting using anti-NR serum indicatedthat irradiation induced the increase in the NR-polypeptidecontent and NO₂ fumigation inhibited the increase, suggestingthat NO₂ put an inhibitory effect on the synthesis of NR inducedby irradiation. ⁴ Present address: College of Environmental Health, Azabu University,Fuchinobe, Sagamihara, Kanagawa 229, Japan ⁵ Present address: Faculty of Home Economics, Otuma Women'sUniversity, Sanban-cho, Chiyoda, Tokyo 102, Japan (Received October 21, 1987; Accepted January 13, 1988)     

Changes in nicotinamide nucleotide content and glucose metabolism in wheat embryos during vernalization

Contents of nicotinamide nucleotides, NAD, NADP, NADH₂ and NADPH₂,in wheat embryos were determined during normal germination andcold treatment. They increased markedly in the early stage ofgermination then decreased later. During cold treatment, thesenucleotides increased initially as in normal germination, butdid not decrease in the later stages. The C₆/C₁ ratios during germination decreased gradually withaging, but after about the fourth day increased. This suggeststhat the pentose phosphate pathway operates actively. Duringcold treatment, the C₆/C₁ ratio decreased slightly with aging.This suggests that the EMP pathway operates predominantly inthe glucose metabolism. Based on these results, the correlationbetween NADPH₂ and glucose metabolism during cold treatmentwas discussed. Embryos which had absorbed labelled glucose were fractionatedinto several chemical components. The radioisotope accumulatedmainly in the sugar fraction, with small amounts in the organicacid, amino acid and nucleic acid fractions. Changes in thecontent of each component showed that sugar, organic acid andamino acid increased during cold treatment. ¹ Present address: Department of Biochemistry, Faculty of PharmaceuticalScience, Higashi Nippon Gaguen University, Ishikari-Tobetsu,Hokkaido 061-02, Japan. ² Present address: Hokkaido Forest Products Research Institute,Asahikawa 070, Japan. ³ Present address: Kakuto 534, Komae City, Tokyo 182, Japan. (Received November 1, 1976; )     

Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

Donation of Electrons to Plastoquinone by NAD(P)H Dehydrogenase and by Ferredoxin-Quinone Reductase in Spinach Chloroplasts

The reduction of plastoquinone by NADPH was detected as an increasein the dark level of Chi fluorescence in osmotically rupturedchloroplasts of spinach. This activity was observed only whenthe chloroplasts were ruptured in a medium containing a highconcentration of MgCl₂. The activity was suppressed by inhibitorsof the respiratory NADH dehydrogenase (NDH) complex in mitochondria,capsaicin and amobarbital, suggesting that the activity wasmediated by chloroplastic NDH complex. Antimycin A, an inhibitorof ferredoxin-quinone reductase (FQR), and the protonophorenigericin also inhibited the increase in Chi fluorescence byNADPH. By contrast, JV-ethylmaleimide (NEM), an inhibitor offerredoxin-NADP⁺ reductase (FNR), did not suppress the fluorescenceincrease, showing that FNR is not involved in this reaction.When the osmotically ruptured chloroplasts were washed by centrifugation,a further addition of ferredoxin as well as NADPH was requiredfor an increase in fluorescence. This ferredoxin-de-pendentactivity also was suppressed by antimycin A, but only partlyinhibited by capsaicin or amobarbital, suggesting that thisis mediated mainly by FQR. These findings suggest that the NADPH-bindingsubunit of NDH complex is easily dissociated from the thylakoidmembranes during the process of the washing the thylakoids bycentrifugation. ³Present address: Shanghai Institute of Plant Physiology, AcademiaSinica, 300 Fenglin Road, Shanghai 200032, China ⁵Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima, 729-02Japan     

An effective time for CO2 gas treatment in overcoming self-incompatibility in Brassica

An investigation was made to determine the effective time forCO₂ treatment in overcoming self-incompatibility in Brassica.CO₂ was effective when supplied to a self-pollinated flowerwhile hundreds of pollen grains were germinating on the stigma.Since the effective time coincides with the attachment of pollentubes to papilla cells, it is thought that CO₂ produces a metabolicchange in these cells during attachement. ¹Part of a thesis submitted for the Dr. of Agr. degree by thesenior author at Tohoku University. ²Present address: Faculty of Agriculture, Kobe University, Nada-ku,Kobe, Japan. (Received December 7, 1972; )     

The metabolism of L-[6-14C]ascorbic acid in detached grape leaves

Grape leaves (Vitis labrusca L.) that are removed from the positionopposite the flower cluster either 28 or 14 days before anthesiscleave L-ascorbic acid (AA) at the C4-C5 bond into a C₄ and,presumably, a C₂ fragment. Leaves taken from this position 14days after anthesis fail to cleave AA. The C₄ fragment is utilizedfor L(+)-tartaric acid (TA) biosynthesis while the C₂ fragmentis recycled into hexose and products of the hexose metabolism.When [6-₁₄C]AA is the source of the label, the sucrose-derivedglucose from labeled leaves has a distribution of ₁₄C in thecarbon skeleton as follows: Cl, 35%; C2, 14%; C3, 4%; C(4+5),13% and C6, 34%. The effect of inhibitors of the glycolate pathwayon [6-^l4C]AA metabolism is examined. ¹This work was supported by Grant No. GM-22427 from the NationalInstitute of General Medical Science, National Institute ofHealth, United States Public Health Service, Bethesda, Maryland.This is Scientific paper No. 5305, Project 0266, College ofAgricultural Research Center, Washington State University, Pullman,WA 99164, U.S.A. ²Present address: The Radioisotope Research Center, Kyoto University,Kyoto 606, Japan. (Received August 29, 1979; )     

A Mutant of Arabidopsis thaliana That Defines a New Locus for Glycine Decarboxylation

A novel photorespiratory mutant of Arabidopsis thaliana, designatedgld2, was isolated based on a growth requirement for abnormallyhigh levels of atmospheric CO₂. Photosynthetic CO₂ fixationwas inhibited in the mutant following illumination in air butnot in atmosphere containing 2% O₂. Photosynthetic assimilationof ¹⁴CO₂ in an atmosphere containing 50% O₂ resulted in accumulationof 48% of the soluble label in glycine in the mutant comparedto 9% in the wild type. The rate of glycine decarboxylationby isolated mitochondria from the mutant was reduced to 6% ofthe wild type rate. In genetic crosses, the mutant complementedtwo previously described photorespiratory mutants of A. thalianathat accumulate glycine during photosynthesis in air due todefects in glycine decarboxylase (glyD, now designated gld1)and serine transhydroxymethylase (stm). Because glycine decarboxylaseis a complex of four enzymes, these results are consistent witha mutation in a glycine decarboxylase subunit other than thataffected in the gld1 mutant. The two gld loci were mapped tochromosomes 2 and 5, respectively. ³Present address: Department of Crop and Soil Sciences, MichiganState University, East Lansing, MI 48824, U.S.A. ⁴Present address: Department of Applied Bioscience, Facultyof Agriculture, Hokkaido University, Kita-Ku, Sapporo, 060 Japan ⁵Present address: Department of Biology, Carnegie Institutionof Washington, 290 Panama Street, Standford, CA 94305, U.S.A.     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )     

Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

The effects of plant growth regulators were investigated onanthocyanin synthesis induced by removing auxin from carrotsuspension cultures. Of the auxins tested, 2,4-D showed thestrongest inhibiting effect on anthocyanin synthesis and hadthe strongest promoting effect on undifferentiated growth. When2,4-D was added to anthocyanin synthesizing cells, in whichcell division had ceased, anthocyanin synthesis was repressedimmediately, accumulated anthocyanin disappeared and cell divisionresumed. All cytokinins examined promoted anthocyanin synthesisin the absence of auxin. Both gibberellic acid (GA₃) and abscisicacid inhibited anthocyanin synthesis in media lacking 2,4-D,though GA³ showed no effect on cell division. These effectsof growth regulators on anthocyanin synthesis are similar tothose reported for their effects on embryogenesis [Fujimuraand Komamine (1975) Plant Sci. Lett. 5: 359, (1979) Z. Pflanzenphysiol.95: 13, (1980) Z. PJlanzenphysiol. 99: 1]. The relationshipbetween the induction of anthocyanin synthesis, metabolic differentiation,and embryogenesis are discussed. ¹ Present address: Department of Biology, College of Arts andSciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo153, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted July 23, 1986)     

Studies on anaerobic biphasic growth of a denitrifying bacterium, Pseudomonas stutzeri

Growth of Pseudomonas stutzeri(VAN NIEL strain) in the presenceof a limiting amount of nitrate under anaerobic conditions ischaracterized by 2 logarithmic phases separated distinctly byan intermediate phase where the growth rate is very low. Inthe first logarithmic phase nitrate is reduced stoichiometricallyto nitrite stage, and in the second phase nitrite is reducedto nitrogen gas. The nitrite reducing activity of cells in the second growthphase is 3–4 times higher than that of cells in the firstphase. The rise in nitrite reducing activity is correlated witha remarkable increase in the content of cytochromes a₂ and c-552. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan. ²Present address: Institute of Molecular Biology, Faculty ofScience, Nagoya University, Nagoya, Japan. (Received June 16, 1969; )     

Amarinin: A New Growth Inhibitor from Luffa amara

A growth inhibitor, amarinin was isolated from the seeds ofLuffa amara. Its formula has been established as 2-deoxy cucurbitacinB by spectrometric analysis coupled with chemical degradation.Amarinin inhibited the growth of the second leaf sheath of riceboth in the absence and presence of GA₃. ¹Present address: Department of Chemistry, Arambag College,P.O. Arambag, Hooghly, West Bengal, India ²Present address: Department of Chemistry, Calcutta University,92, A.P.C. Road, Calcutta 700 009 (Received March 15, 1985; Accepted May 1, 1986)     

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; )     

Lipid-Linked Desaturation of Palmitic Acid in Monogalactosyl Diacylglycerol in the Blue-Green Alga (Cyanobacterium) Anabaena variabilis Studied in Vivo

The mechanism of desaturation of palmitic acid in monogalactosyldiacylglycerol in Anabaena variabilis was studied by labelingin vivo with ¹³C and mass spectrometry. When the cells werefed with [¹³C]Na₂CO₃ for 2.5 h, 19% of the palmitic, but virtuallynone of the palmitoleic, acid at the C-2 position of the lipidwas enriched with ¹³C. During subsequent incubation for 7.5h, the [¹³C]palmitic acid was desaturated to [¹³C]palmitoleicacid. Mass spectrometric analysis of the 2-acylglycerol moietyof the lipid indicated that [¹³C]palmitoyl-[¹³C]glycerol and[¹²C]palmitoyl-[¹²C]glycerol were converted to [¹³C]palmitoleoyl-[¹³C]glyceroland [¹²C]palmitoleoyl-[¹²C]glycerol, respectively. These resultssuggest that the palmitic acid was converted to palmitoleicacid in vivo by lipid-linked desaturation but not via a pathwayconsisting of deacylation, desaturation and reacylation. ⁴Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan ⁵Present address: Department of Physiological Chemistry andNutrition, Faculty of Medicine, University of Tokyo, Hongo,Tokyo 113, Japan (Received December 7, 1985; Accepted April 16, 1986)     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Membrane Potentials in Excitable Cells of Aldrovanda vesiculosa Trap-Lobes

The resting membrane potential in excitable cells of Aldrovandatrap-lobes is composed of diffusion and electrogenic potentials.The diffusion potential, about –100 mV in artificial pondwater, was determined from the external K⁺ and Na⁺ concentrations.The permeability ratio, P_Na/P_K of the membrane was estimatedto be about 0.3. The electrogenic potential hyperpolarized themembrane to about –140 mV. The peak value of the actionpotential increased by +26 mV with a tenfold increase in theexternal Ca²⁺ concentration. The action potential was blockedby an application of the Ca²⁺ chelater or the Ca channel blocker,LaCl₃. Cells showed additional Ca²⁺ influx (7.8 pmole/cm² impulse)during membrane excitation. These facts suggest that the transientincrease in Ca²⁺ influx causes the action potential presentin cells of Aldrovanda trap-lobes. ¹ Present address: Jerry Lewis Neuromuscular Research Center,School of Medicine, University of California Los Angeles, LosAngeles, CA90024, U.S.A. ² Present address: Biological Laboratory, Kyoritsu Women's University,Hachioji 193, Japan. (Received September 21, 1983; Accepted September 7, 1984)     

Evidence for Chlorophyll RC I in Cyanobacteria

We proved the occurrence of chlorophyll RC I (Chl RC I) in theprocaryotic cyanobacteria Anacystis nidulans and Anabaena variabilisand demonstrated its identity with Chl RC I of eucaryotic organismsby spectroscopy and mass spectrometry. The molar ratio of ChlRC I to P-700 was again shown to be unity. It was concludedthat RC I is part of PS I of all organisms with oxygenic photosynthesis. ¹ Present address: Department of Botany, Faculty of Science,Kyoto University, Kita-Shirakawa, Kyoto 606, Japan. (Received May 7, 1985; Accepted September 18, 1985)