LIGHT-INDUCED OXYGEN EVOLUTION CAUSED BY HYDROGEN PEROXIDE IN CATALASE-INHIBITED CHLORELLA CELLS

1. Chlorella cells and spinach chioroplasts, whose catalase activityhad been more than 90% inhibited by 10^–5 M azide, werefound to decompose H₂O₂ photochemically to liberate oxygen,indicating that H₂O₂ was used as an oxidant of the HILL reaction.
2. That, however, the observed phenomena cannot be fully accountedfor in terms of the HILL reaction with H₂O₂ was revealed bythe observation that an extract of Chiorella cells, which hadbeen completely freed from chlorophyll, also showed a light-acceleratedO2 evolution from H₂O₂ in the presence of 10^–5 M azide.This extract contained a large quantity of catalase, which seemedto have been, in some way, involved in the reaction in question.
3. The catalatic H₂O₂ decomposition caused by crystalline catalaseof mammalian liver (in the presence of 10^–5 M azide) wasnot accelerated by the effect of light.

¹ Present address: Department of Biology, Faculty of Science,Niigata University, Niigata. (Received June 4, 1961; )     

PHOTOOXIDATION REACTIONS BY CHLOROPLASTS SOLUBILIZED WITH SURFACE-ACTIVE AGENTS

1. Solubilization of chioroplasts with a mixture of 1 per centDuponol C and 1 per cent Span 80 (3: 1) caused a destructionof activity in the HILL reaction, but the treatment broughtabout an increase by about 60 per cent in the rate of ascorbatephotooxidation in the presence of DPIP. Heating the broken chloroplastscaused a marked decrease in the photooxidation activity. Byadding surface- active agents to the boiled preparation, theactivity was restored up to almost 80 per cent of the originallevel.
2. With colloidal suspensions of isolated chiorophylls,ascorbatewas only slightly photooxidized in the presence ofDPIP. Byaddi tion of the surface-active agents, the activitywas greatlyenhanced.
3. Dependency of the photooxidation bywhole and solubilized chloroplastsand isolated chlorophylla on the presence of DPIP was examined.DPIP can serve as anintermediate electron carrier in solubilizedchloroplasts aswell as in whole chloroplasts.
4. Effect of o-phenanthrolineon ascorbate photooxidation by thesethree preparations wastested. With solubilized chloroplastsand isolated chlorophylls,the addition of the inhibitor hadno influence on their ascorbatephotooxidation either in thepresence or absence of DPIP.
5. Treatmentof whole chloroplasts with the surface-active agentsinducedan activity of photooxidation of cytochrome c. The electron-flowpattern for the photooxidation of ascorbate by whole and solubilizedchloroplasts was briefly discussed.

¹ Contribution No. 130 from the Department of Biology, Facultyof Science, Kyushu University. Aided in part by Grant-in-Aidfor Fundamental Scientific Research from the Ministry of Education. (Received August 23, 1962; )     

FLUORESCENCE YIELD OF CHLOROPHYLL A AND PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS

1. Effect of various treatments on the fluorescence yield of chlorophyllo in isolated chloroplasts was studied in relation to the activitiesof the HILL reaction and the ascorbate photooxidation.
2. CMUand o-phenanthroline showed no appreciable effect on thefluorescenceyield at the concentration which completely inhibited the HILLreaction.
3. 0.01% SDS was sufficient to inactivate the HILLreaction completelyand caused a 40% decrease in the fluorescenceyield, althoughno shift of the absorption and fluorescencebands could be observed.At higher concentrations of SDS, thefluorescence yield increasedby more than three times the originalvalue and the absorptionand fluorescence bands shifted by 5rap toward the shorter wavelengthside.
4. Ultraviolet irradiation,incubation at pH 9.2, heating at 40and digestion with trypsinresulted in loss of the HILL activityand decrease of the fluorescenceyield.
5. The DPIP-mediated photooxidation of ascorbate was enhancedbyheating broken chloroplasts at 55 for 5 min, and inactivatedat 70. The photooxidation of ascorbate in the absence of thedye was inhibited by 60% on heating at 55. The o-phenanthroline-sensitivephotooxidation of ascorbate was inactivated at 55. The o-phenanthroline-resistantphotooxidation of ascorbate was rather thermostable. The fluorescenceyield was reduced by 40–50% at 55.

¹ Present address: Biological Laboratory, General EducationDepartment, Kyushu University, Otsubo-machi, Fukuoka.     

Effects of xylopine on physiological activities of Scenedesmus obliquus

Xylopine, an aporphine alkaloid contained in the bark of Xylopiadiscreta strongly inhibits growth of Scenedesmus obliquus; 50%inhibition was caused by 2x10^–5 M of xylopine. Photosynthesis was also strongly inhibited by xylopine at concentrationsabove 10^–4 M. Effects on photosynthesis, however, differeddepending on the ratio of alkaloid concentration to cell concentrationin the reaction mixture, so that under certain experimentalconditions with concentrations of xylopine below 10^–4M, there was some acceleration of photosynthesis. Xylopine strongly inhibited the HILL reaction with benzoquinoneas the HILL oxidant. Respiration was not affected at concentrationscompletely inhibiting photosynthesisd growth of the alga. A probable role of the aporphine alkaloid in controlling lichengrowth in nature is discussed. (Received March 25, 1970; )     

EFFECT OF LIPASE-DIGESTION ON PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS

In order to elucidate the role of lipids in photosynthesis,chloroplasts were digested with lipase, and the effect of lipase-digestionon some photochemical activities was studied. The HILL reactionwas sensitive to the digestion, but chloroplasts having intactmembrane were somewhat resistant to the action of lipase. Theinactivation by lipase digestion seems to be due to the destructionof a component necessary for the Hill reaction to proceed. Thechloroplasts treated with lipase showed the following activities. (1) Active photooxidation of reduced cytochrome c and menadione. (2) Photooxidation of ascorbate, which was enhanced in the presenceof DPIP, and retarded in the absence of the dye. (3) NADP-photoreduction in the presence of the DPIP-ascorbatecouple, as the electron donor. These facts suggested that the site attacked with lipase wasresponsible for the photochemical oxygen evolution. The decrease in the fluorescence intensity of chlorophyll awas also observed during the digestion. ¹Present address : Biological Laboratory, General EducationDeparment, Kyushu University, Otsubo-machi, Fukuoka.     

Effects of chloride ion on HILL reactions in Euglena chloroplasts

Effects of Cl^– and other anions on the rate of HILL reactionin Euglena chloroplasts were investigated. Cl^– acceleratedthe reaction rate with ferricyanide as HILL oxidant; Br^–,F^– and I^– were also effective; NO₃^–, PO₄^2–and SO₄^2– were less effective. Divalent cations, Ca²⁺and Mg²⁺, were also highly effective. The promoting effectsof these ions were highly dependent on pH and the nature andconcentration of the HILL oxidant used. Accelerating effectsof the ion increased with decreasing concentrations of ferricyanide.Generally, the stimulating effect of Cl^– was much moremarked at pH 7–7.5, with little effect at pH 5. Thus,the pH-activity relationship in the HILL reaction is more orless markedly modified by addition of ions. Cl^–, and other anions, accelerated the reaction by affectingonly the dark rate-limiting portion of the HILL reaction; thelight reaction constant remained uninfluenced. We inferred thatsome reaction step, at which ferricyanide receives electronfrom photosystem 2, is accelerated by Cl^– and other ions.Cl^– effects were rather small, or undetectable, with DPIPor p-benzoquinone as oxidants. (Received January 8, 1970; )     

LIGHT-INDUCED REDUCTION AND OXIDATION OF PLASTOCYANIN BY CHLOROPLAST PREPARATIONS

In the light the intact chloroplasts of spinach reduce plastocyaninwith a reaction rate comparable to that of the usual HILL reaction,while no reduction nor oxidation of the copper protein is inducedby the chloroplasts in the dark. The dependency of the rateof photoreduction upon light intensity, pH and presence of variousreagents was similar to that of the HILL reaction with the usualHILL oxidants. The photoreduction of plastocyanin was acceleratedby the addition of complete phosphorylating system or ammoniumsulfate to the chloroplasts. Digitonin-treated chloroplast wasfound to be inactive in photoreducing plastocyanin but highlyactive in photo-oxidizing reduced plastocyanin. The rate ofphotooxidation was saturated at about 5, 000 lux, and showeda rather broad pH optimum around pH 8.0—8.5. The effectsof various poisons on the reaction rate were studied. When thedigitonin-treated chloroplasts were fractionated with ethanolinto a chlorophyll-bearing participate fraction and a solublefrac tion, the former was active in catalyzing the photooxidationof reduced plastocyanin, but not in photooxidizing reduced cytochromec.An ap preciable photooxidation of reduced cytochrome cwiththe ethanol-precipi tated fraction was obtained on additionof the soluble fraction, which was effectively replaced by plastocyanin.The properties of the reaction systems responsible for photooxidationof plastocyanin and cytochrome C were compared, and a possiblerole of plastocyanin in the photooxidatory process of the chloroplastswas suggested. ¹A part of the present investigation was supported by a researchgrant (GAMN 6208) from ROCKEFELLER Foundation. ²Present address; C.F. KETTERING Research Laboratory, YellowSprings, Ohio, U.S.A.     

The Hill reaction and oxygen uptake in isolated pine chloroplasts

1. The Hill reaction and oxygen uptake in chloroplasts preparedfrom pine leaves were studied. Pine chloroplasts active forthe Hill reaction were isolated from mature leaves in the presenceof 25% PEG in the isolation buffer. Time courses of the Hillreaction in chloroplasts isolated from leaves at different seasonsdiffered. In chloroplasts isolated in the autumn, Hill activitydecreased rapidly with illumination time. This rapid decreaseof Hill activity was inferred to result from concomitant productionof some inhibitory substance(s) during the Hill reaction.
2. Theprotective effect of PEG on inactivation by aging of pinechloroplastswas found. In the presence of PEG, chloroplastswere stabilizedand Hill activity was maintained even afterstorage for 26 hr;whereas, in the absence of PEG inactivationby aging proceededrapidly and oxygen uptake occurred after20 hr.
3. Chloroplastsisolated without PEG had no ability of the Hillreaction; but,inversely showed pronounced oxygen uptake. Oxygenuptake wasalso observed in aged or DCMU-inhibited chloroplasts.The presenceof benzoquinone strongly suppressed oxygen uptake.

¹ Present address: Laboratory of Biology and Chemistry, Universityof Naval Architecture of Nagasaki, Nagasaki, Japan. (Received January 27, 1971; )     

NITRATE REDUCTASE IN PHOTOSYNTHETIC BACTERIUM, RHODOSPIRILLUM RUBRUM. ADAPTIVE FORMATION OF NITRATE REDUCTASE

1. A method was discovered for adapting the cells of Rhodospirillumrubrum to grow on a nitrate medium, a capacity initially lackingin the organism. The adapted cells were able to grow with nitrateas the sole source of nitrogen. The growth responses of theadapted cells towards various nitrogenous sources were investigatedunder various conditions of incubation (aero- and anaerobiosis,light and dark).
2. The adapted cells were found to have simultaneouslyacquiredthe capacity for reducing nitrite and hydroxylamineas wellas nitrate. The path of nitrogen in the adapted cellswas assumedto be as follows: NO₃^– NO₂^– NH₂OH CellularNitrogen.
3. Nitrate metabolism of the adapted cells was investigatedundervarious conditions. In the light, nitrate was reducedand furtherassimilated, leaving insignificant amounts of nitritein themedium. In this case, consumption of nitrate was markedlyinhibitedby other forms of nitrogen (e.g., nitrite, hydroxylamine,aminoacids and ammonium salts). In the dark, nitrate was reducedas the terminal hydrogen acceptor in the oxidative breakdownof organic substances (e.g., malate) in the medium (i.e., nitraterespiration). More nitrite was accumulated in this case thanin the light. Molecular oxygen inhibited the reduction of, aswell as the growth on, nitrate in any of the above cases.
4. Theeffects on the rate of nitrate reduction (and respiratoryoxygenuptake) caused by various experimental factors (pH, nitrateconcentration, electron donors, and addition of hydroxylamine)were investigated, using the resting cells of the adapted organism.

¹ This paper was submitted to the University of Tokyo to fulfillthe requirement for the author's doctorate. ² Present Address: Botanical Institute, Kyoto University, Sakyo-ku,Kyoto. (Received February 14, 1963; )     

COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

DEVELOPMENT OF PHOTOSYNTHETIC ACTIVITIES DURING THE PROCESS OF CHLOROPLAST FORMATION IN CHLORELLA PROTOTHECOIDES

1. By growing Chlorella protothecoides in a medium rich in glucoseand poor in nitrogen source (urea), entirely chlorophyll-lesscells, called "glucose-bleached’ cells, were obtained.These cells were found to have neither discernible plastid structuresnor photosynthetic activities. When these cells were incubatedin a nitrogenenriched mineral medium without added glucose,a remarkable formation of fully organized chloroplasts occurredin the light and only partially organized chloroplasts weredeveloped in darkness.
2. In the dark-incubated algal cells asmall but appreciable amountof chlorophyll was formed, beingaccompanied by developmentof significant activities for thePMS- and FMN-catalyzed photophosphorylationsand the HILL reaction.The development of the capacity for performingphotosyntheticCO₂-fixation, however, was negligible.
3. During the processof "re-generation" of chloroplasts in thelight, there occurredactive formation of chlorophyll followedby development of allthe photic activities mentioned above.Chlorophyll formationas well as development of the photic activitiesproceeded firstin a manner of autocatalytic reaction and laterin the formof the first-order reaction. It was inferred thatthe light-absorbingagent which mediates the chlorophyll synthesisis chlorophyllitself.
4. The activities for the PMS- and FMN-photophosphorylations,theHILL reaction and photosynthetic CO₂-fixation were recognizedalready in the algal cells at an early stage of greening inthe light, in which the "discs" were developed but no completelamellar structure was observed. Further processes of increaseof these photosynthetic and related activities—as measuredat a high and a lower light intensities—were studied inrelation to the chlorophyll formation under continuous illuminationand under light-dark conditions. It was found that the PMS-photophosphorylationactivity was developed always in parallel with the chlorophyllformation under these different light conditions. Developmentof the activities for the other photic reactions, however, lagged,to different extents, behind the formation of chlorophyll inthe later phase of greening of algal cells under these conditions.
5. Based on these results the modes of formation of the componentsinvolved in these photic reactions were surmised.

(Received September 15, 1965; )     

Biological activity of phyllosinol, a phytotoxic compound isolated from a culture filtrate of Phyllosticta sp.

1. Phyllosinol is a phytotoxic metabolite of Phyllosticta sp. Thissubstance at 100 µg/ml produced dark grey necrotic lesionson the leaf of red clover. Sensitivities of various plant speciesto phyllosinol differed both quantitatively and qualitatively.
2. Phyllosinol reduced root growth in rice seedlings by 60% at10^–4 M, whereas stimulation of root elongation occurredat a concentration range from 10^–9 to 10^–5 M.
3. Phyllosinolat 2.5x10^–4M promoted adventitious root formationin epicotylsof Azukia cuttings by about 100%. Promotion waspartly reducedby simultaneous application of cysteine.
4. IAA-induced elongationof isolated Avena coleoptile sectionswas inhibited by phyllosinolat a concentration range from 10^–5to 10^–3M.
5. Sulfhydrylcompounds, i.e. cysteine and glutathione relievedinhibitioncaused by phyllosinol in IAA-induced elongation ofAvena coleoptilesections.
6. GA₃-induced elongation of wheat leaf sections wasslightly inhibitedby phyllosinol at 10^–4M.
7. Phyllosinolalso has antibiotic activity. Among the organismstested, Phycomycetesand Gram-negative bacteria appeared mostsusceptible to phyllosinol.

(Received April 21, 1970; )     

FLAVOPROTEIN IN CHLORELLA ELLIPSOIDEA CATALYZING TPNH-LINKED REDUCTION OF PLASTOCYANIN

1. An enzyme possessing a capacity of catalyzing reduction of thecopper protein, plastocyanin, with reduced pyridine nucleotides(TPNH-plastocyanin reductase) was isolated from Chlorella ellipsoidea.The procedures of purification and the properties of the purifiedenzyme are described.
2. From the results of chromatographicand enzymic tests, the prostheticgroup of the enzyme was identifiedas FAD. No evidence was obtainedto indicate the participationof metal ions in the reaction.
3. The enzyme utilizes both TPNHand DPNH as electron donors, butthe reaction is about 12 timesfaster with TPNH than with DPNH.
4. The enzyme, with TPNH aselectron donor, catalyzes the reductionof Chlorella plastocyanin,cytochrome c, 2,6-dichlorophenolindophenol and oxygen in adecreasing order of reaction rate.The reaction with oxygenas electron acceptor was found to bemuch more strongly acceleratedby the addition of higher concentrationsof flavins as comparedwith the reaction with other acceptors.FAD and FMN added tothe reaction mixture are not appreciablyreduced.
5. The propertiesof the enzyme are compared with those of alliedchloroplastenzymes reported by various investigators and thepossible roleof the new enzyme in photosynthesis is discussed.

(Received January 18, 1961; )     

Light-induced changes of b-type cytochromes in the electron transport chain of Euglena chloroplasts

Light-induced changes of b-type cytochromes in Euglena chloroplastswere studied spectrophotometrically.

1. In the dark and at pH 6.5, most of the cytochrome 558 in chloroplastswas in the reduced state, and most of the cytochrome 563, inthe oxidized state. Illumination of chloroplasts at pH 6.5 induceda rapid, but slight oxidation of cytochrome 552 and cytochrome558. The magnitude of photooxidation of cytochrome 558 was greatlyenhanced by the addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea(DCMU). The rate of photooxidation in the presence of DCMU wasstimulated by the addition of 0.15 µM Euglena cytochrome552, or 100 µM methyl viologen.
2. Euglena chloroplasts,incubated at 55°C for 5 min showedno significant absorbancechanges for about 10 min after theonset of illumination. However,greater photooxidation of cytochrome558 was observed afterprolonged illumination, or in the presenceof DCMU or ethylenediaminetetraaceticacid (EDTA). Similar resultswere obtained with chloroplastspre-treated at pH 9.0–10.0for 5 min.
3. At pH 9.5, andin the dark, both cytochrome 563 and cytochrome558 were inan almost reduced state. On illumination at thispH, both cytochromeswere photooxidized, with a complicatedkinetics, showing aninitial rapid and small absorbance decrease,followed by a stagnantphase of temporary retarded reaction.In the presence of DCMUor EDTA, photooxidation proceeded rapidlywithout a stagnantphase.
4. At pH 6.5 cytochrome 558, on cessation of illumination,wasquickly reduced to the initial level. At pH 9.5, there wasalsoappreciable re-reduction of cytochrome 558 and 563 whenthelight was turned off at an early stage of illumination.Theamounts of re-reduction of the cytochromes in the subsequentdark period, however, decreased as photooxidation of cytochromesproceeded. This decrease was accelerated by the presence ofDCMU.
5. At pH 9.5 ascorbate and manganese served as electrondonorsfor die DCMU-sensitive photooxidation of cytochromes558 and563.
6. Experimental results are discussed with specialreference tothe occurrence of two pools of electron carriers,one at thereducing side and the other at the oxidizing sideof photosystem2. The role of manganese in the latter pool ofelectron carriersis also discussed.

(Received March 11, 1970; )     

RESTORATION OF PHOTOCHEMICAL ACTIVITY OF ISOLATED CHLOROPLASTS LOST BY ALUMINA TREATMENT

Isolated spinach chloroplasts were ground with alumina in amortar with the addition of 0.2 M KC1 solution. This treatmentresulted in a loss of fifty to ninety per cent of the originalphotochemical activity. A petroleum benzin extract was prepared from the alumina residue.After evaporating the solvent from the extract, the residuewas dissolved in methanol. The methanol was evaporated and theresidue was suspended in distilled water and centrifuged. Thecolorless supernatant solution exhibited a reactivation effectwhen added to the alumina-treated chloroplasts. It was inferred from the difference spectrum obtained betweenits oxidized and reduced forms that the active substance inquestion is functioning as intermediary hydrogen carrier inthe HILL reaction. ¹ Contribution No. 100 from the Department of Biology, Facultyof Science, Kyushu University. (Received July 19, 1960; )     

CHANGES IN RESTING POTENTIAL AND ION ABSORPTION INDUCED BY LIGHT IN A SINGLE PLANT CELL

1. Effect of light on ion absorption and resting potential of theinternodal cell of Nitella flexilis was investigated under variousconditions.
2. On illumination, the resting potential increasedby about 30mVin 10^–4 M KCl and by about 60 mV in 10^–4M NaClsolution. A similar photoelectric response was also observedin 10^–3 M KCl, 10^–2 M CaCl₂ and 5 x 10^–2 MCaCl₂ solutions, but not at all in 10^–2 M KCl solution.
3. Absorption of ions by the cell took place in parallel withthelight-induced change in resting potential.
4. Red and bluelights were very effective in increasing the restingpotential,while green light was almost ineffective. These differenteffectsof color lights were in good agreement with their effectsinincreasing the osmotic value of the cell.
5. The photoelectricresponse was not affected by phenylurethane,which, on the otherhand, strongly inhibited the light-inducedion absorption.
6. Theuptake of ions by the cell from the external medium intothevacuole is assumed to proceed in two different steps: thefirstis the process involving the ion movements across theoutermostplasmalemma, and the second is that involved in thetransportof ions through the cytoplasmic layer and tonoplast.The formerprocess is considered to be influenced by the increasein restingpotential probably caused by the light absorbed bychlorophyll.The process was, however, suggested to be independentof photosynthesis.On the other hand, the latter process issupposed to be relatedto photosynthesis. A discussion was madealong this line.

(Received July 26, 1962; )     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

FORMATION OF MYO-INOSITOL AND PHYTIN IN RIPENING RICE GRAINS

With the view to elucidate the role of myo-inositol in the ripeningprocess of rice grains, its distribution, formation and conversionwere studied.

1. myo-Inositol in the ripening rice grains was fractionated intofree-, phosphate ester- and phosphoinositide-forms. At the earlystage of ripening, a considerable part of myo-inositol was foundin free state, and at the end of ripening stage the most partwas found in phosphate ester-state, phytic acid. The contentof phosphoinositide in the grains was low during the ripeningperiod.
2. The occurrence of biosynthesis of myo-inositol inthe ripeningrice grains was confirmed by the observation ofincorporationof ¹⁴C into myo-inositol from ¹⁴C-sugars and itwas found, fromthe feeding experiment of myo-inositol- thatmyo-inositol doesnot undergo reactions further than phosphorylation.
3. The feeding experiment of glucose-l-³²P showed that the distributionpattern of ³²P in different fractions of grain material wasthe same as that of ³²P-phosphate, indicating that phytic acidis one of the final products of phosphorus metabolism in theripening rice grains.
4. These results led to the assumptionthat myo-inositol mightact as an acceptor of phosphorus toremove inorganic phosphorusin favor of starch synthesis byphosphorylase.

(Received September 12, 1962; )     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM II. THE SYSTEM OF CO2-FIXATION IN THIOBAC1LLUS THIOOXIDANS

1. In the early stage of CO₂-fixation by Thiobacillus thiooxidans,which was incubated aerobically in the presence of sulfur, mostpart of the fixed carbon was found in the phosphate ester fraction.
2. The fixation was inhibited by NaF, picolinic acid, PCMB, azide,dipyridyl, o-phenanthroline, monoiodoacetic acid, and arsenite,each in the concentration range where the sulfur oxidation wasnot affected strongly.
3. The crude extract of this organismcould fix CO₂ in the presenceof ATP, R-5-P and Mg⁺⁺. Most partof the fixed carbon was foundin PGA.
4. The crude extract showedthe CO₂-fixation coupled with the H₂S-oxidationin the presenceof ADP.
5. An appreciable reduction of PGA could not be detectedin thepresence of reducing systems, involving TPNH and DPNH.

(Received March 6, 1962; )     

INTERACTION BETWEEN HELIANGINE AND PYRIMIDINES IN ADVENTITIOUS ROOT FORMATION OF PHASEOLUS CUTTINGS

1. Heliangine at 110^–4 M promoted the adventitious rootformation in hypocotyls of cuttings taken from light-grown (1,900lux) Phaseolus mungo seedlings. The promotion was almost completelyreversed by 310^–4 M uracil, uridine, cytidine, oroticacid or 610^–4 M carbamoyl DL-aspartic acid, and partlyby 310^–4 M thymine or thymidine. Neither 310^–4M cytosine, adenine, adenosine, guanine, guanosine nor a combinationof 310^–4 M carbamoyl phosphate and 310^–4 M L-asparticacid reduced the promotion by heliangine.
2. Uracil did not reducethe inhibiting effect of heliangine onthe indoleacetic acidinduced elongation of etiolated Avenacoleoptile sections.
3. Helianginein an aqueous uracil solution was recovered unchangedafter24-hr incubation at room temperature.
4. The root formation ofPhaseolus cuttings was promoted also by2-thiouracil and 5-fluorouracil.The effect was reversed byorotic acid or carbamoyl asparticacid, but not by carbamoylphosphate plus aspartic acid.
5. Ribonucleaseat 100 µg/ml increased the number of rootsprotruded fromhypocotyls of cuttings by about 260%.
6. A possible interpretationfor the promotion of root formationby heliangine is offered.

¹ Contribution No. 15 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. ² Dedicated to Prof. Dr. H. SODING in commemoration of the 70thbirthday.