The Photometabolism of Acetate by Chlorella pyrenoidosa

Chlorella pyrenoidosa can utilize sodium acetate as a carbonsource for growth in the light. Growth proceeds under aerobicconditions both in the presence and in the absence of carbondioxide, but under anaerobic conditions only in its presence.The assimilation of acetate does not result from oxidation tocarbon dioxide followed by photosynthetic fixation because theproducts of ¹⁴C-acetate assimilation are different from theproducts of ¹⁴CO₂ fixation in the presence of unlabelled acetate. In aerobic conditions 10-⁶ M DCMU induces a pattern of acetateassimilation in the light similar to that in the dark. Thus,in the presence of DCMU in the light, less acetate carbon isincorporated into cells, particularly into lipids, polysaccharide,and protein, and more is released as carbon dioxide than inits absence. The effect of 4 x 10^-3 M MFA on acetate assimilationin the presence of 10^-6 M DCMU is the same in light and dark.Acetate assimilation is unaffected by desaspidine and sodiumbisulphite. The mean generation time of C. pyrenoidosa growing on acetatein the light under aerobic conditions is 20 hours. When 10^-5M DCMU is added the mean generation time is 60 hours, the sameas that for Chlorella growing on acetate in the dark. The activityof the enzymes of the glyoxylate cycle, isocitrate lyase (E.C.4.1.3.1.)and malate synthetase (E.C.4.1.3.2.) is repressed in the light,but activity of both enzymes increases markedly when DCMU isadded.     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Photophosphorylation in intact algae: Effects of inhibitors, intensity of light, electron acceptor and donors

The luciferin-luciferase method was used to determine ATP extractedfrom darkmaintained and light-exposed samples of the green algaChlorella pyrenoidosa and of the blue-green alga Anacystis nidulans.A few measurements on Synechococcus lividus (a bluegreen thermophile,clone 65?C) are also reported.

1. The light-minus-dark ATP levels (ATP) from aerobic cells ofChlorella and Anacystis were negative; however, ATP from Synechococcuswas positive. Large positive ATP was obtained in regularly grown(RG: moderate light) Chlorella treated with oligomycin; darklevels were reduced, light levels remained essentially unaffected.In high-light exposed (HLE) Chlorella, oligomycin reduced bothlight and dark ATP levels, but positive ATP was still obtained.However, in Anacystis, which has a different organization ofthylakoid membrane, oligomycin severely reduced both the lightand the dark ATP levels and the ATP remained negative.
2. Theoligomycin (12 µM) treated Chlorella and the untreatedAnacystis and Synechococcus show the presence of cyclic photophosphorylationunder conditions in which the non-cyclic electron flow fromphotosystem II to photosystem I is blocked by 10 µM 3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU), or not allowed to operate by the absence of CO₂. Cyclicphotophosphorylation ranged from 10–30% of the maximumATP in RG, to 40–50% in HLE Chlorella. In RG Chlorella,cyclic and non-cyclic (in the absence of DCMU) photophosphorylation(ATP) saturate at about 10³ ergs cm^–2 sec^–1 and10⁴ ergs cm^–2 sec^–1 and 10⁴ ergs cm^–2 sec^–1red (>640 nm) light, respectively; a lag was observed inthe light curve.
3. In Chlorella, the addition of the photosystemI electron acceptormethyl viologen (MV; 1 mM) increased ATPby twofold. Furtheraddition of DCMU (25 µm) reduced thisto the level observedwith DCMU alone. If 1 mM reduced dichlorophenolindophenol orphenazine methosulphate (DCPIPH₂ or PMSH₂, respectively)wasadded along with DCMU, the ATP level was 30–40% ofthecontrol. Further addition of MV increased the JATP to be70–80%of that of the control. These and other resultsconfirm thepresence of both non-cyclic and cyclic photophosphorylationin vivo, the former predominating in Chlorella, and the latterin Anacystis and Synechococcus.

(Received May 1, 1973; )     

Uptake and Utilization of Fructose by Anabaena variabilis ATCC 29413. Effect on Respiration and Photosynthesis

Anabaena variabilis ATCC 29413 showed a constitutive mechanismfor fructose uptake which was further enhanced by growing thecells with fructose. The uptake process was energydependentas indicated the inhibitory effect of the uncoupler carbonylcyanidem-chlorophenylhydrazone (CCCP) and the reduction induced byincubating the cells in the dark or in the light with DCMU.Cells adapted to growth on fructose showed increased rates ofrespiration both in the dark and in the light. The rate of ¹⁴CO₂evolved from radiolabelled fructose was lower in the light thanin the dark or in the presence of DCMU. This fact can be partiallyexplained by the photosynthetic reutilization of respiratory¹⁴CO₂. Modifications in photosynthesis were observed in fructose-growncells. PS I and PS II activity measured in spheroplasts obtainedby lysozyme treatment were enhanced by fructose probably asa way to compensate the lower concentration of chorophyll showedby fructose-grown cells. The photosynthetic affinity for externalCO₂ and the rate of photosynthesis dependent on external inorganiccarbon were reduced by fructose. (Received September 24, 1991; Accepted February 14, 1992)     

Role of pulvinar chloroplasts in light-driven leaf movements of the trifoliate leaf of bean (Phaseolus vulgaris L.)

Laminar pulvini of bean (Phaseolus vulgaris L.) contain numerouschloroplasts in cells of their motor tissue. The quantitativerelationships of the chloroplast pigments, chlorophyll a andb, ß-carotene, lutein, neoxanthin as well as the xanthophyllcycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin)were similar to those of mesophyll chloroplasts from leafletlaminae. Exposure of pulvinules to light caused deepoxidationof violaxanthin to zeaxanthin, showing that the xanthophyllcycle is functioning. Chlorophyll fluorescence analysis of pulvinulesconfirmed that their chloroplasts are capable of both photosyntheticelectron transport and non-photochemical fluorescence quenching,showing that they build up a considerable transthylakoid protongradient in the light. Application of DCMU to excised pulvinulesand laminar discs, as well as to pulvinules of intact, attachedterminal leaflets blocked electron transport and fluorescencequenching. Application of the uncoupler CCCP to intact pulvinulesalso prevented non-photochemical fluorescence quenching. Therate of movement of the low-light-adapted terminal leaflet inresponse to exposure of its pulvinule to overhead red light(500 µmol m^–2 s^–1) was reduced when the pulvinulewas pretreated with DCMU. The pulvinar response to overheadblue light (50 µmol ^–2 s^–1), which is morepronounced than to red light, was not affected by similar pretreatmentwith DCMU. Pretreatment with CCCP caused a short lag in theresponse to red light, but did not affect its subsequent rate.The results suggest that the pulvinar response to red, but notto blue light, requires non-cyclic electron transport and theresulting generation of ATP Key words: Leaf movements, light, non-cyclic electron transport, Phaseolus, pulvinar chloroplasts     

Short-term Products of 14C-acetate Assimilation by Chlorella pyrenoidosa in the Light

The kinetics of ¹⁴C-2-acetate assimilation by Chlorella pyrenoidosain the light were examined. Under aerobic conditions the primaryproduct of acetate assimilation was succinic acid which, afterten seconds, contained over 60 per cent of the ¹⁴C incorporatedby the cells. The percentage of the total ¹⁴C in succinate fellwith time, while that in citrate and glutamate increased. After1800 sec over 60 per cent of ¹⁴C was present in two compounds,glutamic acid and an unknown compound (X). Glucose-6-phosphate,fructose-6-phosphate, phosphoglyceric acid and phosphoenolpyruvicacid became labelled after 60 sec but together never containedmore than one per cent of the total ¹⁴C incorporated. Underanaerobic conditions succinate was still the primary productof acetate assimilation, and the absence of carbon dioxide resultedin a decrease in ¹⁴C incorporation into compound X. The patternof acetate assimilation in acetate grown and acetate adaptedChlorella was very similar to that in photo-autotrophicallygrown Chlorella. In the presence of 10^–6M DCMU, succinicacid was the primary product of acetate assimilation, but therewas an early Incorporation of ¹⁴C into glutamate, aspartate,and malate. 4 x10^–3M MFA did not effect the early incorporationof ¹⁴C into succinic acid, but resulted in accumulation of ¹⁴Cin citrate and a decreased amount in glutamate and in compound X.     

Studies on the Cell Wall of Chlorella IV. Incorporation of Glucose-Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

Uniformly ¹⁴C-labeIled glucose was fed to synchronously growingChlorella cells in the dark or in light. The rate of ¹⁴C-incorporationinto hemicellulose showed two maxima one in the growth phaseand one in the reproductive phase. Significant ¹⁴Cincorporationinto a "rigid wall" was found only in the reproductive phase. (Received April 14, 1983; Accepted June 15, 1983)     

Effect of High-intensity Light Given Prior to Low-temperature Treatment on the Long-day Flowering of Pharbitis nil

Pharbitis nil, strain Violet which had been exposed to high-intensitylight (18,000 lux at 23?C) for 7 days followed by a low-temperaturetreatment (13–14?C) for 7 days initiated flower buds evenunder continuous light, but plants given these treatments inreverse order failed to bud. Three days of high-intensity lightat 23?C was most effective in promoting the flower-inducingeffect of the subsequent low-temperature period. Six days oflow temperature following the 3-day high-intensity light periodinduced near-maximum flowering response. DCMU (5?10^–6M) given during the high-intensity light period inhibited flowering,but when given during or after the low-temperature period itwas ineffective. DCMU at the same concentration given before,during or after an inductive 16-hr dark period at 26?C did notinhibit flowering. Sucrose, ATP, NADPH and some other reducingagents tested did not nullify the DCMU effect nor substitutefor the effect of high-intensity light. But, the high-intensitylight effect could be substituted, at least partly, by 5-chlorosalicylicacid, 3,4-dichlorobenzoic acid and some other benzoic acid derivatives,which are highly effective in inducing long-day flowering inthe short-day plant, Lemna paucicostata. (Received October 20, 1981; Accepted February 3, 1982)     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Effects of disalicylidenepropandiamine and near far-red light on 14CO2-fixation in Chlorella cells

1) With Chlorella ellipsoidea cells, in the presence of 5x10^–6M DSPD, or in its absence, the amounts of ¹⁴CO₂ incorporatedin P-esters, serine-plus-glycine and alanine were larger underred light than under blue light, whereas blue light specificallyincreased ¹⁴CO₂-incorporation in aspartate, glutamate, malateand fumarate (blue light effect). The amount of total ¹⁴C fixedunder blue or red light was greatly decreased by the additionof DSPD. When the concentration of DSPD was raised to 5x10^–4M, practically no radioactivity was found, under blue or redlight, in aspartate, glutamate and fumarate. Radioactivity inalanine was greatly increased. Effects of higher concentrationof DSPD are explained as due to the inhibition of PEP carboxylaseactivity in Chlorella cells. 2) The percentage incorporation of ¹⁴C into aspartate and theother compounds mentioned above, under near infra-red illuminationwas significantly smaller than that under blue light and wasalmost equal to that under red light. These results along withthe effect of 5x10^–6 M DSPD, exclude the possibility thatcyclic photophosphorylation is involved in the "blue light effect"mechanism. (Received December 12, 1969; )     

Photodestruction of Pigments in Higher Plants by Herbicide Action: I. THE EFFECT OF DCMU (DIURON) ON ISOLATED CHLOROPLASTS

3-(3',4'-Dichlorophenyl)-1',1'-dimethyl urea (DCMU) inducedthe photobleaching of chlorophylls and carotenoids in isolatedchloroplasts of Hordeum vulgare. In chloroplasts illuminatedin both the absence and presence of DCMU (5.0 mmol m^–3),the destruction of carotenoid preceded that of the chlorophylls.The rate of photodestruction was accelerated by the presenceof DCMU. After only 2 h illumination the rates of loss of ß-caroteneand of the epoxyxanthophylls, neoxanthin and violaxanthin, weresimilar (approximately 40–50% loss in the presence of5–0 mmol m^–3 DCMU) but weremuch greater than thatof lutein (25% loss). Analysis of the individual pigment-proteincomplexes, isolated from chloroplasts following such treatment,showed that whilst pigment destruction had occurred in all complexes,the relative content of the LHCP2/CPa complexes (containingthe PSII core) had fallen to the greatest extent. Further illuminationof the chloroplasts, for up to 22 h, resulted in far greaterbleaching but showed a similar pattern of pigment loss, withDCMU again accelerating the rate at which this loss occurred.ß-Carotene-5,6-epoxide was identified as a productof such photo-oxidative conditions. Key words: DCMU, carotenoids, chlorophylls, photobleaching, ß-carotene-5,6-expoxide     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Metabolism of Glycerolipids in Plastidial and Extraplastidial Membranes of Etiolated Wheat Leaves: In Vivo and In Vitro Studies

Etiolated wheat leaves were fed with [l-¹⁴C]-acetate and chaseexperiments were performed in the dark or under light. In bothconditions, in extraplastidial membranes, phosphatidylglycerol(PG) and phosphatidylcholine (PC) were fatty acid donors, PGand PC providing palmitate and oleate respectively. The labelof linoleate increased only in phosphatidylethanolamine (PE).In etioplasts, PG was also a palmitate donor but PC, ratherpoor in labelled oleate, was an oleate acceptor, contrary towhat was observed in chloroplasts. The galactolipids and sulfoquinovosyldiacylglycerol(SL) remained poorly labelled. When isolated etioplasts were labelled in vitro, during thefirst two hours they incorporated the same amount of [l-¹⁴C]-acetatein their phospholipids, whether they were in the presence orin the absence of extraplastidial membranes. Afterwards, theaddition of a mitochondrial fraction enhanced the label of PG,mainly in palmitate, then in oleate, and to some extent, andonly under light in palmitoleate and linoleate. The mitochondrialfraction might be regarded here as a supplier of labelled precursorto he etioplasts which rapidly accumulated radioactivity inpalmitoyl-PG. In PC of isolated etioplasts, only palmitate wasfairly labelled. The deficiency in labelled oleoyl-PC in plastidsof dark-grown plants and of linoleoyl-PC in extraplastidialmembranes might be the reason for the delay in the labellingof unsaturated galactolipids. ¹ (Received March 9, 1987; Accepted August 21, 1987)     

Glycerolipid Synthesis in Avena Leaves during Greening of Etiolated Seedlings IV. Effect of Light on Fatty Acid Desaturation

Etiolated Avena sativa L. leaves were fed with [l-¹⁴C]acetatefor 20 h in the dark and labeled fatty acids in glycerolipidswere chased during 24 h in the light (greening condition) orin the dark, to determine the light effect on the fatty aciddesaturation. Oleate decrease in phosphatidylcholine was thesame in the light and in the dark, showing that oleate desaturationis independent of light (or greening). Linoleate desaturationin galactolipids, especially in monogalactosyl diacylglycerol,was enhanced by light and palmitate desaturation to hexadecenoatein phosphatidylglycerol was strictly light-dependent. (Received May 11, 1983; Accepted August 16, 1983)     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )     

Studies on frost hardiness in Chlorella ellipsoidea III. Changes in O2 uptake and evolution during hardening and after freeze-thawing

Chlorella ellipsoidea cells at an intermediate stage in theripening phase of the cell cycle were hardened at 3?C. Oligomycin(OGM) and 3-(3,4-dichiorophenyl)-1,1-dimethylurea (DCMU) addedduring hardening in the light inhibited the development of frosthardiness, suggesting that mitochondria and chloroplasts wereinvolved in the hardening process. The O₂-uptake activity in unhardened cells increased duringhardening in the light while the O²-evolution activity decreased,when these activities were measured at 25?C. The increase inO₂ uptake was suppressed by OGM and DCMU and the decrease inO₂ evolution was stimulated by OGM. While the algal hardinessin the dark was very limited, the addition of glucose duringhardening in the dark caused a remarkable development of frosthardiness. These results suggest that mitochondria and chloroplastsclosely interact at low temperature, and the former plays aprincipal role in the hardening process and the latter servesas substrate-donor in the light. The O₂ evolution in cells which survived freezing was remarkablydecreased by freeze-thawing while the O₂ uptake was hardly affected.The freeze-injured chloroplasts were repaired during the followingincubation. OGM inhibited the repair of freeze-injured chloroplasts.From the results, mitochondria seem to change their membranesinto a structure hardier than chloroplasts, and ATP synthesizedby mitochondria seems to be essential for the repair of freeze-injuredchloroplasts. ¹ Present address: Department of Public Health, Faculty of Medicine,Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka 812, Japan. (Received November 9, 1977; )     

Photosystem I and photophosphorylation-dependent leucine incorporation in the blue-green alga Anacystis nidulans

L-Leucine uptake and incorporation in the blue-green alga Anacystisnidulans were measured during illumination with monochromaticlight of 630 and 717 nm. With near as well as far red light,an enhanced uptake of ¹⁴C-L-leucine was observed. In far redlight, the leucine uptake depended on light intensity and pHvalue. After the first few minutes, the uptake remained constantfor more than one hour. The rate of uptake in light was thesame in air as in nitrogen. The incorporation of ¹⁴C-leucinein the soluble fraction decreased in the presence of chloramphenicolwhich prevents protein synthesis. In far red light, its incorporationwas insensitive to DCMU (5 ? 10^–6 M) but was depressedby uncouplers like CCCP or desaspidin. These effects are takenas evidence that leucine incorporation under the conditionsused is dependent on photosystem I reactions and cyclic photophosphorylation.DBMIB and KCN in high concentrations decrease the leucine incorporationin far red light and indicate that plastoquinone and plastocyaninare members of the cyclic electron flow also in intact cellsof Anacystis. Antimycin A has no inhibitory effect. The inhibitionby other less specific inhibitors like salicylaldoxime, desaspidinand DSPD is discussed. (Received August 19, 1978; )