ROLE OF NUCLEUS IN CYTOPLASMIC ACTIVITIES WITH SPECIAL REFERENCE TO THE FORMATION OF SURFACE MEMBRANE IN ELODEA LEAF CELLS

Using leaf cells of Elodea densa, investigations were made onthe role of nucleus in some activities of protoplasm, by comparingthe behaviours of nucleated and enucleated halves of protoplast,which were separated by means of plasmolytic treatment of intactcells.

1. Plasmolytic treatment caused a remarkable chloroplast-systrophe,which was more pronounced in nucleated halves than in enucleatedones, in which the systrophe, if any, was dispersed gradually.
2. In general, protoplasmic streaming was suppressed under plasmolyticconditions, although in nucleated protoplasm a rotation of systrophedchloroplast-bunch was often observed after a longer durationof culture.
3. The membrane which was re-formed on the surfaceof divided protoplasmichalves was found to be more solidifiedand less elastic in thenucleated halves than in the enucleatedhalves.
4. Detection of RNA in the cytoplasm by UNNA-PAPPENHEIM'sstainingmethod showed that the ability of pyronin uptake wasdecreasedin the enucleated halves as compared with that inthe nucleatedhalves.
5. By treatment with ribonuclease, theproperties of nucleatedprotoplasmic halves were rendered tobe similar to those ofthe enucleated halves.
6. Based on theobservations, discussions were made on the r?leof nucleus inthe regulation of the membrane formation by, andthe viscosityand other physico-chemical properties of, thecytoplasm.

¹ This study was supported by a grant-in-aid for DevelopmentalScientific Research from the Ministry of Education, No. 407041(1955), and No. 407084 (1960). (Received November 19, 1960; )     

CHLOROPHYLL-SENSITIZED REDUCTION OF PLASTOQUINONE BY ASCORBIC ACID AND QUENCHING OF CHLOROPHYLL-FLUORESCENCE BY PLASTOQUINONE

1. Photochemical reduction of plastoquinone by ascorbic acid inethanol was sensitized with some derivatives of chlorophyll.The order of effectiveness was as follows: allomerized chlorophylla > chlorophyllin a > chlorophyll a > chlorophyll b> pheophytin a.
2. Quenching of the fluorescence of chlorophylla by plastoquinonewas observed. The quenching constant calculatedwas 71 litreper mole.

¹Contribution No. 159 from the Department of Biology, Facultyof Science, Kyushu University. Supported in part by a grant-in-aidfor Fundamental Scientific Research from the Ministry of Education. ²Present address: Biological Laboratory, General Education Department,Kyushu University, Ropponmatsu, Fukuoka. ³Present address: Biological Institute, Daiichi College of PharmaceuticalSciences, Tamagawa-machi, Takamiya, Fukuoka.     

PHOTOSYNTHETIC NITRITE REDUCTASE FROM SPINACH

Photosynthetic nitrite reductase (PNiR) was purified extensivelyfrom spinach leaves by the method including acetone precipitation,ammonium sulfate fractionations and DEAE-cellulose column chromatography.The purified PNiR had the highest specific activity thus farobtained. Absorption spectrum of the purified PNiR indicatedthat it had no flavin component. There was no difference inthe rate of photosynthetic nitrite reduction between aerobicand anaerobic conditions. In chloroplasts two reductase systems related with ferredoxinhad been recognized, which were ferredoxin-nitrite and ferredoxin-NADPreductase system. The relationship between the two systems wasinvestigated by using the reconstituted enzyme system. Whenthe reduction of NADP was taking place, the reduction of nitritewas strongly inhibited. Some physiological significance of thephenomenon was discussed. ¹This work was mainly perfomed in the Department of Cell Physiology,University of California, during the author's stay, and waspresented in the 6th annual meeting of the Japanese Societyof Plant Physiologists on April 4-7, 1964 in Tokyo.     

ENZYMIC FORMATION OF GLUTAMINE IN RICE-PLANT SEEDLINGS

1. Purified preparation from rice-plant seedling catalyses a stoichiometricreaction between ATP, glutamate, and NH₂OH in the presence ofMg⁺⁺ to form glutamyl hydroxamate, ADP and inorganic phosphate.
2. The method of purification and some of the properties of theenzyme are described. Co⁺⁺ can be substituted for Mg⁺⁺. Mn⁺⁺,NaF, and PCMB inhibit the enzyme strongly.
3. Inorganic orthophosphateis liberated from ATP by the additionof or cysteine in the presence of glutamate, Mg⁺⁺ andthis preparation. Glutamine was detectedin the reaction productsby paperchromatography.
4. The same preparation catalyses a reactionbetween gluta mineand NH₂OH in the presence of Mg⁺⁺ or Mn⁺⁺,ADP and inorganicphosphate, to form glutamyl hydroxamate.

¹ Present address: The Department of Chemistry, Faculty of Science,Kanazawa University, Kanazawa. (Received October 31, 1960; )     

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; )     

WATER PERMEABILITY OF THE CELL WALL IN NITELLA

1. A method has been developed to measure the hydraulic conductivityof the wall of the internodal cell of Nitella flexilis.
2. Therate of water penetration through the cell wall varies linearlywith the hydrostatic pressure difference between the two sidesof the wall, showing that water permeability of the cell wallremains independent of the pressure difference applied.
3. Waterpermeability of the cell wall is inversely proportionalto itsthickness It is 30µµmin^–3{dot}atm^–3when the thickness of the wall is 10 µ.
4. Water permeabilityof the cell wall is the same for inward andoutward water flow.The polar water permeability of the entiremembrane system (walland protoplasmic part) of the living celldemonstrated by KAMIYAand TAZAWA (1) is, therefore, due tothe living protoplasmicpart.
5. The ratio of the inward to outward permeability constantsofthe protoplasmic layer alone is higher than that of the entiremembrane system composed of protoplasmic layer and cell wall.

¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.The present work was supported in part by a Grant-in-Aid forFundamental Scientific Research from the Ministry of Education. ² Present address: Sh?in Women's College, Kobe. (Received July 21, 1962; )     

Purification and properties of the photoactive particle corresponding to photosystem II

1. 1) Purification of the photoactive particle corresponding tophotosystem II (particle II) was achieved using a combinationof procedures including digitonin- and Triton X-100-treatments,sonication, and differential- and density-gradient centrifugations.
2. 2) The "purified" particle II preparation showed only oxygenevolution activity and showed no NADP⁺ photoreduction activityeven when an electron donor couple was added.
3. 3) The chemicalcompositions of this particle and of the particlecorrespondingto photosystem I (particle I) were compared withrespect tothe composition and contents of their chlorophylls,carotenoids,cytochromes, plastoquinones, protein and lipid.Marked differenceswere found.
4. 4) Using the two photoactive particles I and IIlacking therespective counterpart activity, a partial successwas attainedin reconstituting a system, in which photoinducedelectron flowfrom water to NADP⁺ was observed, provided thatsuitable intermediateelectron carriers, e. g. plastoquinoneand plastocyanin, wereadded.
5. 5) The nature of the two photoactiveparticles obtained andtheir relationship to particles so farreported are discussed.

¹This study was aided by grants from the Ministry of Education(407160-1965, 91402-1966, 1967). Financial support from SanyoBroadcasting Scientific Foundation is also acknowledged withcordial thanks. (Received October 23, 1968; )     

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; )     

STUDIES ON A CAROTENE-PROTEIN COMPLEX ISOLATED FROM GREEN LEAVES

1. Two new methods of preparing a carotene-protein complex fromchloroplasts were described, in addition to the one previouslyreported by the present authors.
2. Light-scattering measurementswere performed and from the dissymmetryratio of the scattering,the particle size of the complex wascalculated. The mean radiuscorresponding to the weightaverageparticle weight was foundto be 56 mµ. This result wasdiscussed with referenceto the previously reported values obtainedfrom sedimentationmeasurements and electron micrographic determinations.
3. Thedepolarization of scattered light was measured. Large valuesobtained for the degree of depolarization of the incident lightrevealed a high degree of anisotropy in the particle of thecarotene-protein complex.

¹Present address: Johnson Foundation, University of Pennsylvania,Philadelphia, Pa., U.S.A. (Received July 8, 1960; )     

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; )     

STUDIES ON CHLOROPHYLLASE OF CHLORELLA PROTOTHECOIDESI. ENZYMATIC PHYTYLATION OF METHYL CHLOROPHYLLIDE

1. The relation between chlorophyll content and the hydrolyticactivity of chlorophyllase in Chlorella protothecoides was examined.An increase in the activity was parallel to that in chlorophyllcontent during the development of green colouration, or greeningcourse, in the bleached cells. The activity sharply declinedand a parallel disappearance of chlorophyll was also found duringbleaching of the green cells.
2. A partially purified water-solublepreparation of chlorophyllasewas obtained by n-butanol treatmentand fractionation with coldacetone. It showed high activityand hydrolyzed 2 mg chlorophylla per hr per mg protein.
3. Forseparation and identification of the pigments concernedin thechlorophyllase reaction, a new solvent system of paperchromatographywas introduced.
4. When methyl chlorophyllide a and phytol wereincubated withthe enzyme, two products were formed. By comparisonwith theRf values of isolated pure substances, one was identifiedaschorophyll a and the other as chlorophyllide a. This enzymedid not catalyze the phytylation of free chlorophyllide a, butit had the ability to attach phytol to methyl chlorophyllidea. The final step in the biosynthesis of chlorophyll a is brieflydiscussed.

¹ Contribution No. 158 from the Department of Biology, Facultyof Science, Kyushu University. Supported in part by a grant-in-aidfor Fundamental Scientific Research from the Ministry of Education.     

FORMATION OF PHYCOERYTHRIN IN PRE-ILLUMINATED CELLS OF TOLYPOTHRIX TENUIS WITH SPECIAL REFERENCE TO NITROGEN METABOLISM

1. The formation, in the dark, of phycoerythrin in the preilluminatedcells of a blue-green alga, Tolypothrix tenuis, was investigatedwith special reference to its nitrogen metabolism.
2. On incubatingthe pre-illuminated algal cells under a darkaerobiccondition,and with nitrate as N-source, the formation of phycoerythrinoccurs after an induction period of about 5 hours. No time-lagis observed in the nitrate-uptake by the organism. Similar resultsare obtained with nitrite, ammonia, urea and arginine as N-sources.
3. The above stated formation of phycoerythrin is suppressedbysubstances such as chloramphenicol and p-fluorophenylalanine,substances known to be potent inhibitors of protein-synthesis.
4. On the basis of these findings, it was inferred that therearetwo consecutive processes involved in the dark-formationofphycoerythrin in the pre-illuminated cells: (i) uptake andconversionof exogenous nitrogen sources into some intermediarynitrogenouscell substances, and (ii) synthesis of the pigment-proteinfromthese substances.

(Received June 27, 1960; )     

Coupling of malate decarboxylation to CO2 fixation and the reduction of 3-phosphoglyceric acid in corn bundle sheath cells,2

1. In the presence of NADP⁺ and Mg⁺⁺, the bundle sheath strandsisolated from corn (Zea mays) leaves by cellulase treatmentsdecarboxylated malate in the light at an initial rate (200 µmoles/mgchl.hr), which was sufficient to account for photosyntheticCO₂ fixation in intact leaves. This rate gradually slowed downand then stopped. The final level of the malate decarboxylatedwas approximately equal to the amount of NADP⁺ added.
2. Rapidand continued decarboxylation of malate was observed whenNADP⁺,3-phosphoglyceric acid and ATP (and Mg⁺⁺) were addedtogether.The addition of ADP instead of ATP showed a similareffect.Light did not show any effect on the malate decarboxylationin the presence of ATP or ADP.
3. When malate was added to thebundle sheath strands in the presenceof exogenous NADP⁺ NADP⁺was rapidly reduced. The reductionstopped after 2 min when,73% of the added NADP⁺ was reduced.The further addition of3-phosphoglyceric acid and ATP broughtabout a decrease in theNADPH-level, which rose again to attaina new steady level.
4. The transfer of radioactivity from (1-¹⁴C-3-phosphoglycericacid to dihydroxyacetone phosphate in the bundle sheath strandsin the presence of ATP and NADP⁺ was greatly enhanced by theaddition of malate.
5. In the presence of ribose 5-phosphateand ATP, the rate of ¹⁴C-transferfrom (4-¹⁴C)-malate to theintermediates of the reductive pentosephosphate cycle was equalto that of ¹⁴CO₂ fixation in the light.

All these results support the current view that in the bundlesheath cells of C₄ plants belonging to the NADP-malic enzyme-group,the decarboxylation of malate is coupled to the fixation ofthe released CO₂ and the reduction of 3-phosphoglyceric acidformed as a result of CO₂ fixation. ¹ Part of this research was reported at the 40th Annual Meetingof the Botanical Society of Japan Osaka, December, 1975. ³ Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, 359 Otsuka, Hachioji-City, Tokyo 173, Japan. (Received April 30, 1977; )     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION. I.

1. The role of sulfur in the cell division of Chlorella was studiedby following the fate of the sulfur supplied to the sulfur-deficientcells using ³⁵S as a tracer.
2. The sulfur-deficient cells whichwere unable to perform celldivision were made capable of divisionby the provision of ³⁶S-labeledsulfate under non-photosynthesizingconditions. Soon after theprovision of sulfate the labeledsulfur went rapidly into thecold perchloric acid (PCA)-solublefraction of algal cells,almost entirely in the form of sulfateand/or some other inorganicsulfur substance (s). With the lapseof time, more or less remarkablechanges occurred in the patternof ³⁵S-distribution in differentfractions of cell material.It was noticed that, at the onsetof cell division, a sulfur-containingpeptide-nucleotide compound(s)(SPN), which has been reportedearlier, appeared in a largequantity in the cold PCA-solublefraction, and that its quantitydecreased gradually during thesubsequent process of cell division,suggesting that the compoundwas transformed into some othersubstance (s), presumably withits nucleotide moiety going intonucleic acids and the peptidemoiety going into some essentialproteins.
3. Another noteworthyphenomenon observed during the process ofcell division wasthe incorporation of ³⁶S in a group of hotPCA-soluble substances.These sulfur substances were revealedto be sulfur-containingnucleotidic compounds, which might possiblybe some essentialcomponents of, or substances in close relationto, deoxypentosenucleic acid (DNA).

(Received March 1, 1960; )     

PHOTOCHEMICAL REDUCTION AND OXIDATION OF MENADIONE (VITAMIN K3) IN CHLOROPLASTS

1. Menadione (vitamin K³) was found to be completely reduced byilluminated spinach chloroplasts under highly anaerobic conditionand in the presence of ethylenediamine tetraacetate (EDTA) inthe reaction mixture. This photoreductive reaction is sensitivetoward heat-treatment and inhibited by 2?10^-3M hydroxylamine.
2. In the presence of oxygen, the reduced form of menadione israpidly photooxidized by chloroplasts. This photooxidative activityalso is suppressed by heat-treatment but not inhibited by hydroxylamine.
3. Dyes which are inefficient as HILL oxidants such as thionineand methylene blue were found to be readily reduced by illuminatedchloroplasts, if the experimental conditions were appropriateto prevent the reoxidation of the photoreduced dyes; i.e., exhaustiveremoval of oxygen and the addition of EDTA in the reaction mixture.Menadione was found to accelerate the HILL reaction with thesedyes as oxidant under such experimental conditions.
4. In thepresence of molecular oxygen in the reaction mixture,menadionewas found to inhibit the HILL reaction with 2,6–dichlorophenolindophenol as oxidant, while the reaction rate was little influencedin high anaerobiosis.
5. These findings are explained by theintermediary oxidation and(photo-) reduction of menadione asan intermediary hydrogencarrier, and by the trends toward rapidphotooxidation of reducedmenadione.

(Received July 2, 1960; )     

ADAPTIVE FORMATION OF NITRATE REDUCING SYSTEM IN ANABAENA CYLINDRICA

1. Changes in capacities for reducing nitrate, nitrite and hydroxylaminecaused by provision or depletion of various nitrogen sourceswere investigated with a nitrogen fixing blue-green alga, Anabaenacylindrica, and adaptive nature of these reducing system wasdemonstrated.
2. It was found that, under light-aerobic conditions,nitrate-and nitrite- reducing systems were induced by nitrateor nitritebut not by N₂ ammonia and glutamate. On the otherhand, theactivity of enzymes pertaining to hydroxylamine reductionwasstimulated equally by nitrate, nitrite and N₂. The latteractivitywas suppressed markedly in the presence of ammoniaor glutamate.
3. Adaptive formation of nitrite reducing systemis completelyinhibited by chloramphenicol, a potent inhibitorof proteinsynthesis. No formation was also observed under theanaerobiccondition or in the dark.
4. On the basis of thesefindings, a tentative scheme for pathwaysof nitrate reductionand nitrogen fixation in Analaena cylindricawas proposed.

(Received August 22, 1962; )     

Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrical

Nitrite reductase was purified about 40-fold from the blue-greenalga Anabaena cylindrica by acetone precipitation and chromatographyon DEAE-cellulose columns. The nitrite reductase had its pHoptima at about 7.6 with Tris-HCl and at about 7.4 with phosphatewhen reduced methyl viologen was used as an electron donor.The Km's for nitrite, methyl viologen and ferredoxin were 510^–55,210^–4 and 510^–6M, respectively. A stoichiometryof one molecule of ammonia formation per one molecule of nitritedisappearance was confirmed. Ferredoxin which had been reducedeither chemically with dithionite or enzymatically with NADPHin the presence of diaphorase was active as an electron donor.Dithionite-reduced FAD and FMN were inactive. NADPH could notgive electrons directly to nitrite reductase. Hydroxylaminereductase was segregated from nitrite reductase by DEAE-cellulosecolumn chromatography. Purified nitrite reductase showed noactivity for sulfite reduction. A molecular weight of 68,000was estimated for nitrite reductase using a calibrated SephadexG-200 column. ¹This work was supported by grants 4090 and 955008 from theMinistry of Education. ²This work was supported by grants 4090 and 955008 from theMinistry of Education. 2 Present address: Department of Botany,Faculty of Science, University of Tokyo, Tokyo.     

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.     

Photosynthetic and light-enhanced dark fixation of 14CO2 from the ambient atmosphere and 14C-bicarbonate infiltrated through vascular bundles in maize leaves

1. The capacity of light-enhanced dark fixation of ¹⁴CO₂ from theambient atmosphere decayed following time-course characteristicsof a first-order reaction (half-life, 1–2 min). The levelof phosphoenolpyruvate in maize leaves under CO₂-free air didnot decrease in the dark subsequent to preillumination. Theseresults indicate that phosphoenolpyruvate carboxylase is activatedin light and quickly inactivated in the following darkness.
2. Removal of oxygen from the atmosphere did not exert any effecton the products of light-enhanced dark fixation of ¹⁴CO₂ providedfrom the atmosphere, the major labeled compounds being malateand aspartate. This confirms that the transfer of carboxyl carbonof C₄-acids to form 3-phosphoglycerate is light-dependent.
3. WhenNaH¹⁴CO₃ solution was vacuum-infiltrated through vasculartissuesof maize leaves, the main initial photosynthetic ¹⁴CO₂fixationproducts were phosphate esters. This indicates thatby thistechnique, ¹⁴CO₂ could be directly provided to the bundlesheathcells, and was fixed via the reductive pentose phosphatecycle.On the other hand, the main initial ¹⁴CO₂-fixation productswere malate and aspartate even when ¹⁴CO₂ was provided throughvascular tissues in the dark immediately following preillumination.The possible regulatory mechanisms underlying the above findingsare discussed.

¹ This work was reported at the 4th International Congress onPhotosynthesis, Reading, September 1977. Request for reprintsshould be addressed to S. Miyachi, Institute of Applied Microbiology,University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ² Present address: Okinawa Branch of Tropical Agriculture ResearchCenter, Ishigaki-shi, Okinawa 907, Japan. (Received October 28, 1977; )     

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.