The Inhibitory Effect of Light on Proton-Coupled Hexose Uptake in Chlorella

The addition of 3-O-methyl-D-glucopyranose to wild-type cellsof Chlorella vulgaris (211-11h) grown in glucose medium in thedark induced a rapid alkalization of the external medium (protonuptake), whereas this pH shift did not occur in autotrophicallygrown cells. Light-irradiation inhibited the sugar-induced protonuptake, making the blue end of the visible spectrum very effectiveand the red end only slightly effective. This spectral dependencecorresponds to that of photoinhibition on hexose uptake in thesealgae. A similar photoinhibitory effect was observed in cellsof a colorless mutant of Chlorella vulgaris (M125). The activity of nitrate-proton symport in Chlorella vulgaris(211-11h) was also enhanced by the addition of glucose. Illuminationhad no inhibitory effect on this increased transport. The effectof light on the hexose uptake system is discussed. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)     

Effects of Light on Growth and Glucose Consumption in Colorless Chlorella Mutant Cells

Light inhibited the growth and glucose consumption of colorlessmutant cells of Chlorella vulgaris (# 125). Sugar consumptionwas also inhibited in a medium containing a hexose such as D-fructose,D-galactose and D-mannose. Blue light strongly inhibited growth and glucose consumptionbut red light only slightly affected them. Respiration was notinhibited by blue light. The inhibitions of growth and glucoseconsumption were saturated at light intensities as low as 800mW.m^–2 and continued in the dark for at least one dayafter brief illumination with white light. The half-maximumeffect was observed with 15 min of illumination in both casesand the action spectra for light-induced inhibitions of growthand glucose consumption were similar, both showing peaks at370, 457 and 640 nm. The role of light in the inhibitions of growth and glucose consumptionis discussed. (Received June 18, 1984; Accepted October 29, 1984)     

Alkalization of the Medium by Unicellular Green Algae during Uptake Dissolved Inorganic Carbon

When Chlorella vulgaris 11h, Chlorella vulgaris C-l, Chlamydomonasreinhardtii, Chlamydomonas moewusii, Scenedesmus obliquus, orDunaliella tertiolecta were illuminated in with 0.5 mM NaHCO₃,the pH of the medium increased in a few minutes from 6 to about9 or 10. The alkalization, which was accompanied by O₂ evolution,was dependent on light, external dissolved inorganic carbon(DIC) as HCO^-₃, and algae grown or adapted to a low, air-levelCO₂ in order to develop a DIC concentrating mechanism. Therewas little pH increase by algae without a DIC concentratingprocess from growth on 3% CO₂ in air. Photosynthetic O₂ evolutionwithout alkalization occurred using either internal DIC or externalCO₂ at acidic pH. The PH increase stopped between pH 9 to 10,but the alkalization would restart upon re-acidification betweenpH 6 and 8. Alkalization was suppressed by the carbonic anhydraseinhibitors, acetazolamide, ethoxyzolamide or carbon oxysulfide.The pH increase appeared to be the consequence of the externalconversion of HCO^–₃ into CO₂ plus OH^– during photosynthesisby cells with a high affinity for CO₂ uptake. Cells grown onhigh CO₂ to suppress the DIC pump, when given low levels ofHCO^–₃ in the light, acidified the medium from pH 10 to7. Air adapted Scenedesmus cells with a HCO^–₃ pump, aswell as a CO₂ pump, alkalized the medium very rapidly in thelight to a pH of over 10, as well as slower in the dark or inthe light with DCMU or without external DIC and O₂ evolution.Alkalization of the medium during photosynthetic DIC uptakeby algae has been considered to be part of the global carboncycle for converting H₂CO₃ to HCO^–₃ and for the formationof carbonate salts by calcareous algae from the alkaline conversionof bicarbonate to carbonate. These processes seem to be a consequenceof the algal CO₂ concentrating process. ¹Present address: Department of Biology, Faculty of Science,Niigata University, Niigata, 950-21 Japan.     

Transport and Fixation of Inorganic Carbon in the Cells of Chlorella vulgaris 11h Grown under Ordinary Air

Intracellular accumulation of inorganic carbon (C_i) and itsfixation in photosynthesis were investigated using siliconeoil layer filtering centrifugation technique with the cellsof Chlorella vulgaris 11h grown under ordinary air. Both CO₂and HCO₃^– were transported into the cells from the reactionmedium and accumulated in the cells, but the rate of transportwas much faster for the former than the latter. ¹⁴C-fixationfrom the total transported C_i was much more efficient when CO₂was added in the external medium than when HCO₃^– was added.This indicates that CO₂ and HCO₃^– were not converted tothe common compound in the cells during the initial period ofphotosynthesis. Accumulation of Cⁱ into the cells was much lesssusceptible to low temperature than its fixation. Accumulationof Cⁱ was also observed in the dark. Ethoxyzolamide, an inhibitorof carbonic anhydrase (CA), inhibited the fixation of accumulatedCO₂ in the cells, suggesting that CA enhanced the supply ofCO₂ to the reaction site of ribulose bisphosphate carboxylasein the stroma. Mechanism for transport and fixation of Cⁱ duringphotosynthesis in low-CO₂ cells of C. vulgaris 1lh was proposedfrom these results. (Received March 19, 1986; Accepted June 26, 1986)     

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; )     

Light-induced Cell Division in Chlorella vulgaris, Beijerinck (Emerson Strain)

The inability of the Emerson strain of Chlorella vulgaris togrow and divide actively in a glucose medium in the dark hasbeen confirmed. It has been shown that although glucose doesnot enhance the rate of cell-division when added to culturesgrowing under saturating photosynthetic conditions, it neverthelessmarkedly increases the growth-rate when supplied to culturesin which photosynthesis is limited by an inadequate CO₂ supply. Transfer of actively growing cultures from light to darknessis followed by a limited period of active cell-division if glucoseis added to the medium; this has been interpreted as indicatingglucose utilization and the synthesis in light only, of somesubstance(s) essential for cell-division. Further evidence forthis view has been obtained from studies of the effect of alight pretreatment on subsequent growth in the dark. With cultures aerated with CO₂-free air, re-exposure to lightafter a period in the dark has been shown to bring about a resumptionof active cell-division accompanied by a decrease in the percentageof ‘giant’ cells in the population. This also suggeststhe participation of some photo-reaction, other than photosynthesis,in the control of active cell-division in this strain of C.vulgaris.     

Effects of blue light on respiration and carbon dioxide fixation in colorless Chlorella mutant cells

A colorless mutant of Chlorella vulgaris (Mutant #125) starvedin darkness, showed suppressed rates of respiration and darkCO₂ fixation, which were significantly recovered by illuminationwith blue light. The main CO₂ fixation product under blue lightwas aspartate. Such enhancements did not take place in cellsactively growing in the glucose medium. Both enhancing effectsof blue light (456 nm) were saturated at light intensities aslow as 400–800 erg.cm^-2.sec^-1. The action spectra forthese enhancing effects were similar to each other; both showedpeaks at 460 nm and 380 nm, which correspond to the absorptionmaxima of flavin. All these findings indicate that the samemechanism underlies the observed effects of blue light on CO₂fixation and respiration. The role of blue light which bringsabout the enhancements in CO₂ fixation and respiration is discussed. (Received June 1, 1974; )     

Photosynthetic metabolism of 14CO2 in the process of the "glucose-bleaching" of Chlorella protothecoides

Changes in photosynthetic carbon metabolism during the glucosebleaching of Chlorella protothecoides cells were investigatedusing NaH¹⁴CO₃ as tracer. Several hours after incubating thegreen algal cells in the glucose medium in the dark, the ratesof ¹⁴C-incorporation into glucose polymers and sucrose decreasedand the incorporation into the lipid fraction (fatty acids)greatly increased. At this stage, the rate of photosynthetic¹⁴CO₂ fixation and the chlorophyll content were practicallythe same as in the starting green cells. Afterwards, the photosyntheticcapacity and chlorophyll content continued to decrease throughoutthe experimental period. In contrast, when photosynthetic ¹⁴CO₂fixation of green cells was carried out in the medium containingglucose, the rate of ¹⁴C-incorporation into glucose polymersincreased, though there was no change in the incorporationsinto sucrose and the lipid fraction. ¹Part of this investigation was reported at the Conference "ComparativeBiochemistry and Biophysics of Photosynthesis" (Japan-U.S. CooperativeScience Program) held at Hakone, Japan in 1967. ²Present address: Faculty of Agriculture, Tamagawa University,Machida-shi, Tokyo, Japan. (Received June 10, 1974; )     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 2. The Fate of Radioactive Carbon

Most of the ¹⁴C added as glucose to carbohydrate-starved cellsof Chlorella Vulgaris can be recovered as alcohol-soluble compoundsor as polysaccharide. Only 5–I6 per cent., depending onthe position of ¹⁴C in the glucose supplied, is released ascarbon dioxide. Similar results were obtained with Chlorellapyrenoidosa and Ankistrodesmus. The labelled alcohol-solublecompounds in Chlorella vulgaris include amino-acids, particularlyglutamic acid, aspartic acid, and alanine, and, when glucose-I-¹⁴Cis metabolized, the amount of ¹⁴C recovered in these amino-acidsis about the same as that recovered as carbon dioxide. Degradationof the glucose incorporated into polysaccharide shown that theC₁ and C₆ atoms of glucose rapidly interchange when in the cells.The bearing of these results on attempts to estimate the relativeimportance of different pathways of glucose breakdown is discussed.     

Wavelength effects on photosynthetic carbon metabolism in Chlorella

To study the wavelength-effect on photosynthetic carbon metabolism,¹⁴C-bicarbon-ate was added to Chlorella vulgaris 1 lh suspensionunder monochromatic blue (456 nm) and red (660 nm) light. Thelight intensities were so adjusted that the rates of ¹⁴CO₂ fixationunder blue and red light were practically equal. Analysis of¹⁴C-fixation products revealed that the rates of ¹⁴CO₂ incorporationinto sucrose and starch were greater under red light than underblue light, while blue light specifically enhanced ¹⁴CO₂ incorporationinto alanine, aspartate, glutamate, glutamine, malate, citrate,lipid fraction and alcohol-water insoluble non-carbohydratefraction. Pretreatment of the algal cells in phosphate mediumin the dark, which was essential for the blue light enhancementof PEP carboxylase activity, was not necessary to induce theabove wavelength effects. Superimposition of monochromatic bluelight at low intensity (450 erg.cm^–2.sec^–1) on thered light at saturating intensity caused a significant decreasein the rate of ¹⁴CO₂ incorporation into sucrose and increasein incorporation into alanine, lipid-fraction, aspartate andother related compounds, indicating that the path of carbonin photosynthesis is regulated by short wavelengdi light ofvery low intensity. Possible effects of wavelength regulationof photosynthetic carbon metabolism in algal cells are discussed. ¹ Part of this investigation was reported at the XII InternationalBotanical Congress, Leningrad, 1975 and the Japan-US CooperativeScience Seminar "Biological Solar Energy Conversion", Miami,1976. Requests for reprints should be addressed to S. Miyachi,Radioisotope Centre, University of Tokyo, Bunkyo-ku, Tokyo 113,Japan. ⁴ Present address: Department of Chemistry, Faculty of PharmaceuticalSciences, Teikyo Univ., Sagamiko, Kanagawa, Japan. (Received August 6, 1977; )     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)     

EFFECTS OF LIGHT ON THE DEOXYRIBONUCLEIC ACID FORMATION AND CELLULAR DIVISION IN CHLORELLA PROTOTHECOIDES

1. It has been demonstrated previously that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), chlorophyll-less cells with markedly degenerated plastids—called "glucose-bleached" cells—are produced eitherin the light or in darkness. When the glucose-bleached cellsare incubated in a medium enriched with the nitrogen sourcebut without added glucose, normal green cells with fully organizedchloroplasts are obtained in the light, and pale green cellswith partially organized chloroplasts in darkness. During theseprocesses of chloroplast development in the glucose-bleachedcells, there occurs, after a certain lag period, an active DNAformation followed by a more or less synchronous cellular division.In the present study the effects of light on the DNA formationand cellular division were investigated in the presence of CMUor under aeration of CO²-free air to exclude the interveninginfluence of photosynthetic process.
2. It was revealed thatlight severely suppresses the DNA formationand cellular divisionof the glucose-bleached cells while enhancingremarkably theirgreening. The suppression was saturated atthe light intensityof about 1,000 lux. Blue light was mosteffective, being followedby green, yellow and red light inthe order of decreasing effectiveness.
3. Further experiments unveiled that light exerts two apparentlyopposing effects on the DNA formation depending upon the timeof application during the incubation of algal cells. When thealgal cells were illuminated only during the lag period beforethe active DNA synthesis, there occurred an enhancement of theDNA synthesis occurring during the subsequent dark incubation.When, on the other hand, the cells were transferred to the lightfrom darkness at or after the start of the DNA synthesis, itcaused an almost complete abolition of the subsequent synthesisof DNA in the algal cells. No such effects of light were observedwith RNA and protein (total)
4. These findings were discussedin relation to the process ofchlorophyll formation occurringconcurrently in the algal cells.

(Received August 10, 1967; )     

Effects of CO2 concentration during growth on subsequent photosynthetic CO2 fixation in Chlorella1

The maximum rate of photosynthetic ¹⁴CO₂ fixation (V_max) aswell as the concentration of CO₂ at which the rate of photosynthetic¹⁴CO₂ fixation attains one-half its maximum velocity (K_m) inChlorella vulgaris 11h cells was strongly dependent on the concentrationof CO₂ continuously provided during the algal growth. The V_max (µmoles ¹⁴CO₂ fixed/ml pcv?min) and K_m (% CO₂)of the algal cells which had been grown in air containing 4%CO₂ (by volume) were ca. 10 and 0.15–0.17, while thosein the cells which had been grown in ordinary air (containing0.04% CO₂) were 7 and 0.05–0.06, respectively. When the concentration of CO₂ in the bubbling gas was loweredfrom 4 to 0.04% during the algal growth, their photosynthetickinetics attained the respective lower steady levels after 5–10hr. On the other hand, when the photosynthetic kinetics weredetermined 24 hr after raising the concentration of CO₂ from0.04 to 4%, the V_max and K_m-values were found to have alreadyattained the respective higher levels. (Received October 15, 1976; )     

Form of inorganic carbon utilized for photosynthesis in Chlorella vulgaris 11 h cells

The rate of photosynthetic ¹⁴CO₂ fixation in Chlorella vulgaris11h cells in the presence of 0.55 mM NaH¹⁴CO₃ at pH 8.0 (20?C)was greatly enhanced by the addition of carbonic anhydrase (CA).However, when air containing 400 ppm ¹⁴CO₂ was bubbled throughthe algal suspension, the rate of ¹⁴CO₂ fixation immediatelyafter the start of the bubbling was suppressed by CA. Theseeffects of CA were observed in cells which had been grown inair containing 2% CO₂ (high-CO₂ cells) as well as those grownin ordinary air (containing 0.04% CO₂, low-CO₂ cells). We thereforeconcluded that, irrespective of the CO₂ concentration givento the algal cells during growth, the active species of inorganiccarbon absorbed by Chlorella cells is free CO₂ and they cannotutilize bicarbonate. The effects observed in the high-CO₂ cellswere much more pronounced than those in the high-CO₂ cells.This difference was accounted for by the difference in the affinityfor CO₂ in photosynthesis between the high- and low-CO₂ cells. (Received May 19, 1978; )     

Diurnal Rb+ Transport from Roots to the Laminar Pulvinus in Phaseolus vulgaris L.

The primary leaves of kidney bean (Phaseolus vulgaris L.) openunder light and close in the dark by the deformation of thepulvinus resulting from diurnal distribution changes of K⁺,Cl^–, organic acid (or H⁺) and NO₃^–. When Rb⁺ was added as a tracer of K⁺ to the seedlings throughtheir roots, it was transported to the pulvinus cells duringthe light period but not during the dark period. Transpirationoccurred vigorously in the light but almost stopped in the dark.We concluded that Rb⁺ absorbed by the roots was carried to thepulvinus by the transpiration stream. Phaseolus vulgaris L., pulvinus, Rb⁺, diurnal transport transpiration stream     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides I. Role of non-photosynthetic CO2-fixation in chloroplast regeneration

Carbon dioxide enhanced chloroplast regeneration in glucose-bleachedcells of Chlorella protothecoides in the presence of CMU inthe light. Both the formation of chlorophyll and the synthesesof RNA and protein were considerably enhanced. The CO₂ metabolism of algal cells during greening was investigatedusing ¹⁴C-bicarbonate as the tracer. Radiocarbon was largelyincorporated into purine and pyrimidine bases in nucleic acidand the arginine in protein, specifically at the crabon atomsderived from carbamylphosphate. ¹Part of this investigation was reported at the conference onthe "Autonomy and biogenesis of mitochondria and chloroplasts"held at Canberra in 1969 (4). (Received August 19, 1975; )     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

Photoinhibition of 5-aminolevulinic acid formation under GO2-free condition

5-Aminolevulinate accumulation in the presence of levulinatewas followed in greening Chlorella protothecoides cells. Underthe CO₂-free condition, ALA formation was severely inhibitedby 20 W/m² white light. The inhibition was removed by CMU. Combinedaddition of CMU with N, N'-tetramethyl phenylenediamine plusascorbate again caused photoinhibition of ALA formation, whilethe addition of CMU with dithiothreitol caused severe inhibitionof ALA formation in both light and darkness. Exogenous glucose enhanced ALA formation in darkened algal celb,but not in photo- and DTT-inhibited cells. In either case, glucoseseemed to be metabolized mainly by the algal cells through theglycolysis-citric acid system. It was inferred that ALA formationwas suppressed at the site of, or related to, an enzyme reactionforming ALA. (Received June 27, 1979; )     

Suppressive effect of light on the formation of DNA and on the increase of deoxythymidine monophosphate kinase activity in Chlorella protothecoides

As previously demonstrated, chlorophyll-less cells of Chlorellaprotothecoides are obtained when the alga is grown in a mediumrich in glucose and poor in a nitrogen source (urea). When thesecells are incubated in a medium enriched with a nitrogen source,there occurs, besides greening of algal cells, an active formationof DNA followed by synchronous cellular division. The DNA formationand cellular division are markedly suppressed by light of acomparatively low intensity. Blue light is most effective andred light least effective in suppression. The effect of light on the level of dTMP kinase activity inthe algal cells was investigated in relation to the photoinhibitionof DNA formation. It was found that light suppresses the increaseof dTMP kinase activity in the algal cells which starts in advanceof active DNA synthesis, and that blue light has a strongersuppressive effect than red light. ¹Present address: Institute of Medical Science, University ofTokyo, Tokyo.     

Light-Induced Changes in the Molecular Size of Starch in Colorless Chlorella Cells

Starch from colorless mutant cells of Chlorella vulgaris (#125), grown heterotrophically in the dark, was fractionatedby agarose gel chromatography. The molecular weight distributionof starch showed only one pronounced maximum at 2?10⁶ (L-starch).Exposure of cells to white light decreased significantly inthe amounts of the total starch and L-starch. This also wastrue under non-growth conditions, the same effect being foundto depend on blue light. The role of light on starch degradationis discussed. (Received November 29, 1984; Accepted February 25, 1985)