CHANGES IN CONTENTS OF CARBOHYDRATE AND FATTY ACID IN THE CELLS OF CHLORELLA PROTOTHECOIDES DURING THE PROCESSES OF DE- AND RE-GENERATION OF CHLOROPLASTS

1. Previous work has demonstrated that when cells of Chlorellaprotothecoides are grown mixotrophically under illuminationin a medium rich in nitrogen source (urea) and poor in glucose,normal green cells are obtained, while in a medium rich in glucoseand poor in the nitrogen source, strongly bleached cells containingapparently no discernible chloroplast structures — called"glucose-bleached" cells — are produced either in thelight or in darkness. When the green cells are incubated ina glucose-enriched mineral medium without added nitrogen source,they are fairly rapidly bleached with concomitant degenerationof chloroplast structures (" bleaching "). When, on the otherhand, the "glucose-bleached" cells are transferred in a nitrogen-enrichedmedium without added glucose under illumination, they turn greenwith regeneration of chloroplasts (" greening "). In the presentstudy changes in contents of carbohydrate and fatty acid inalgal cells were followed during these processes of "bleaching"and "greening.".
2. During the process of "bleaching", the quantityof glucose existingin the insoluble carbohydrate fraction ofalgal cells increasedrapidly and markedly. A considerable increasewas also observedin the contents of cells in oleic, linoleicand palmitic acids.It was noted, however, that linolenic aciddecreased in quantityduring the most active phase of cell bleaching.
3. During the process of "greening", the glucose in the insolublecarbohydrate fraction rapidly decreased, suggesting that itis utilized, as carbon and energy sources, for the chloroplastregeneration. Linolenic acid was found to be synthesized inparallel with formation of chlorophyll. A peculiar pattern ofchange in contents was observed with oleic and palmitic acids,which was interpreted as being related with the process of cellulardivision occurring incidentally during the process of greening.

(Received September 24, 1966; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: II. EFFECTS OF ACTINOMYCIN ON GREENING OF "GLUCOSE-BLEACHED" AND "ETIOLATED" ALGAL CELLS

The "glucose-bleached" and "etiolated" cells of Chlorella protothecoideshaving plastids of different degrees of degeneration were preparedby the methods previously reported, and the effects of actinomycin(C complex) upon the processes of greening of these cells wereinvestigated under various experimental conditions. As has beenshown previously, these cells formed normal chloroplasts onbeing incubated in the light with provision of nitrogen source(urea), but without glucose. The greening process of the glucose-bleachedcells has been found to differ from that of the etiolated cellsin the point that it involves a light-independent phase precedinga light-requiring phase. It was revealed that the greening ofglucose-bleached cells is inhibited by actinomycin much morestrongly than that of etiolated cells. On applying the antibioticat different times during the chloroplast development in glucose-bleachedcells, it was found that the inhibitory effect was remarkablyreduced with the progress of the developmental process. Thisindicated that the antibiotic attacked more strongly the light-independentphase than the light-requiring phase in question. Based on theseobservations it was inferred that, in the process of chloroplastdevelopment in glucose-bleached cells, DNA and RNA are playingimportant roles, especially during the early light-independentphase of chloroplast development. (Received September 18, 1964; )     

RIBONUCLEIC ACIDS APPEARING DURING THE PROCESS OF CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Investigations were made on the modes of synthesis of differentspecies of RNA which appear during the greening (chloroplastregeneration) of the "glucose-bleached" cells of Chlorella protothecoidescontaining profoundly degenerated plastids.
2. RNAs were extractedfrom the algal cells which had been labelledwith ³²P for 1hr before harvesting at different stages of thegreening inthe light and in darkness, and subjected to columnchromatographywith methylated albumin-coated kieselguhr. Itwas found that,during the greening process, the elution profilesof RNAs, interms of the optical density at 260 mµ and³²P-radioactivity,changed profoundly.
3. Based on these and other results, it wasconcluded that duringan early phase of the chloroplast regenerationin the glucosebleachedalgal cells, there occurs an active formationof both ribosomalRNAs (rRNAs) and the RNAs corresponding tosoluble RNA (sRNA),the formation coming, however, later toa standstill when thesynthesis of chlorophyll has proceededto a certain level. Thequantity ratio of sRNA to rRNA was foundto be constant (30:70)at different stages of the greening (bothin the light and indarkness), with a few exceptions. The synthesisof the chloroplastribosomal RNA is markedly accelerated bylight, and its maximumrate is observed sometime later thanthat of the non-chloroplast("cytoplasmic") ribosomal RNA. Itwas suggested that there areat least two different sites ofsynthesis of ribosomal RNAs,one in the plastid and the otheroutside of it (most probablyin the nucleus).

¹A part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965. ² Present address: Institute for Plant Virus Research, Ministryof Agriculture and Forestry, Aoba-cho, Chiba.     

EFFECT OF LIGHT ON THE CHLOROPHYLL FORMATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Previous studies have shown that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), apparently chlorophyll-less cells with profoundly degeneratedplastids—referred to as "glucose-bleached cells—areproduced either in the light or in darkness. When the glucose-bleachedcells are incubated in a medium enriched with the nitrogen sourcebut without added glucose, an active formation of chlorophylloccurs after a certain lag period under illumination, whilein darkness a very small amount of chlorophyll is formed atabout the same time as in the light. The stimulating effectof light on the chlorophyll formation is not appreciably affectedwhen the photosynthetic CO₂-fixation of greening algal cellsis blocked by the addition of CMU. In the present study, itwas further found that the light-enhanced chlorophyll formationproceeds, although at a somewhat lower rate, under aerationof CO₂-free air. All the experiments in this work were doneunder these non-photosynthetic conditions to exclude any influenceof photosynthates.
2. The effect of light (from daylight fluorescentlamps) on thechlorophyll formation in the glucose-bleachedalgal cells wassaturating at about 1,000 lux. Blue light wasfound to be mosteffective; yellow, green and red light followingin the orderof decreasing effectiveness.
3. When the bleachedalgal cells were illuminated for a short periodin the lag phaseof chlorophyll formation and subsequently incubatedin darkness,there occurred an appreciable enhancement of chlorophyllformationin the dark. When the short illumination was appliedat differenttimes of the lag phase, the enhancement was inducedto almostthe same extent. But the longer the duration of theilluminationduring the lag phase, the greater was the enhancementof chlorophyllformation in the subsequent dark incubation.In such experimentsblue light was most effective and red lightleast, as it wasthe case in the experiments of continuous illumination.An intervenientillumination of the bleached cells at lowertemperatures orunder the atmosphere of N2 produced little orno enhancementof the chlorophyll formation in the subsequentdark incubation.
4. Based on these results, it was concluded that the light enhancementof chlorophyll formation in the glucose-bleached algal cellsis mediated by a non-chlorophyllous photoreceptor(s), absorbingmaximally blue and yellow light, and that a light-induced changeof the photoreceptor is immediately followed by a certain dark(temperaturedependent and aerobic) process(es) which is connected,directly or indirectly, to the chlorophyll synthesis.

(Received August 10, 1967; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: III. EFFECTS OF MITOMYCIN C ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

The "glucose-bleached" cells of Chlorella protothecoides, whoseplastids were profoundly degenerated containing no trace ofchlorophyll, were obtained by the method previously reported.Transferring the cells to the condition of re-generation ofchloroplasts (greening)—incubation in the light in a glucose-lessand nitrogen-rich medium—the effect of mitomycin C onthe recovery process was investigated. It was found that theantibiotic suppressed completely the cell division without affectingthe re-generation of chloroplasts. De novo formation of RNAand protein which has been observed to occur during the recoveryprocess was not affected by the antibiotic to any significantextent. It thus became clear that the re-generation of chloroplasts,accompanied by the formation of chlorophyll, RNA and protein,occurring under the said condition is not a phenomenon causedby the formation of new "normal" cells from previously degeneratedcells. As was expected, the antibiotic suppressed strongly theDNA synthesis, indicating that the new formation of DNA is nota necessary condition for the re-generation of chloroplastsin "glucose-bleached" algal cells. (Received March 1, 1965; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: IV. EFFECTS OF 5-FLUOROURACIL, DIHYDROSTREPTOMYCIN, CHLORAMPHENICOL AND ACRIDINE ORANGE ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

1. The "glucose-bleached" cells of Chlorella protothecoides, whichwere obtained by the method described previously, were transferredto a glucose-free medium containing basal mineral nutrientsalone in the dark, and after a certain period of time, the cellsuspension was supplied with urea and light to induce the greeningof cells. At different times before and after the provisionof urea and light, the inhibitors were applied to the cultureto test their effects upon the process of greening.
2. Markedgreening of the glucose-bleached cells occurred aftera lagperiod in the control culture. 5-Fluorouracil inhibitedthecell greening strongly when it was applied at differenttimesbefore the provision of urea and light. When applied aftertheprovision of urea and light, the suppressive effect of 5-fluorouracilgradually decreased with the delay of its application. No inhibitiveeffect was observed when the uracil analogue was added laterthan the 12th hr after the provision of urea and light, thetime around which the chlorophyll formation started in the controlculture. On the other hand, the cell division was much morestrongly affected by 5-fluorouracil. Even when it was appliedat the 18th hr after the provision of urea and light, the celldivision was completely halted, indicating that the greeningand division of the glucose-bleached cells are separate processes.Different mechanisms of action of the uracil analogue towardsthese two processes were suggested.
3. Dihydrostreptomycin showedits strongest suppressive effectwhen added at the beginningof the dark incubation of algalcells in the glucose-free medium,and with the delay of application,its effect was progressivelyreduced, even during the periodof the dark incubation. Thesuppression, however, was stillmarked when it was applied atthe 15th hr.
4. Chloramphenicol was found to inhibit stronglythe chlorophyllformation and protein synthesis, but, to a muchlesser extent,RNA synthesis. Acridine orange suppressed thecell greeningand division at such a low concentration as 1.5µg/ml.
5. Based on these observations it was concludedthat synthesesof nucleic acid and protein are essential processesfor thegreening of the glucose-bleached algal cells. Successiveeventsoccurring in the greening process were discussed.

(Received March 9, 1965; )     

METABOLISM OF GLUCOSE IN THE PROCESS OF "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, normal cells of Chlorella protothecoidesare bleached with degeneration of chloroplasts when they areincubated, under aerobic conditions—either in the lightor in darkness—, in a glucose-containing medium withoutadded nitrogen source ("glucose-bleaching"). It was found inthe present study that under the atmosphere of N₂, neither bleachingnor growth of algal cells occurs in the dark, while in the lighta significant growth of cells takes place with formation ofa certain amount of chlorophyll.
2. Studies on the effects ofvarious inhibitors (ammonium ion,DNP, CMU, -hydroxysulphonates,arsenate, cyanide, azide, andantimycin A) under different conditionsshowed that oxidativephosphorylation is a necessary processfor the occurrence ofthe glucosebleaching as well as the assimilationof glucose(cellular growth). Under light-anaerobic conditionsin the presenceof glucose, assimilation of glucose (cellulargrowth) takesplace being supported by photophosphorylation,but no bleachingoccurs.
3. When the algal cells in the courseof bleaching were transferredto the glucose-free mineral medium,the cell growth ceased immediatelybut the cell bleaching proceededfor several hours before itscessation. The respiratory activity,which was high in the glucose-containingmedium, became loweron transferring the algal cells into theglucose-free medium.The lowered level of respiration was maintained,for more than8 hr after the transfer of cells to the glucose-freemedium.
4. When the cells in the course of bleaching were placed underthe atmosphere of N₂, the cell bleaching ceased almost instantaneously.
5. Based on these observations and other inhibition experiments,it was inferred that a certain intermediate(s) produced by theaerobic respiration of glucose is closely associated with theoccurrence of cell bleaching, and that an O₂-requiring stepmay be involved in the process of chlorophyll degradation.

(Received September 9, 1965; )     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: I. SYNTHESES OF NUCLEIC ACIDS AND PROTEIN IN RELATION TO THE PROCESS OF REGENERATION OF CHLOROPLAST

When Chlorella protothecoides is grown mixotrophically in thelight in a medium rich in glucose and poor in nitrogen source(urea), one obtains the cells that are entirely devoid of chlorophylland containing only little RNA and protein. When these cells—referredto as "glucose-bleached" cells—are further grown in thelight with provision of nitrogen source, but without glucose,sequential syntheses of RNA, protein and chlorophyll take place.If the glucose-bleached cells are incubated in the dark underthe same nutritional condition, RNA, protein and chlorophyllare also successively formed in relatively small amounts. Thecells obtained under such a condition are, in many respects,similar to the cells that are obtained when the alga is grownin the dark in a medium poor in glucose and rich in the nitrogensource. These cells, which are called the "etiolated cells",are faintly green in color and contain larger amounts of RNAand protein compared with the chlorophyll-less glucose-bleachedcells. The glucose-bleached cells and the etiolated cells showapproximately the same content of DNA per cell. When the etiolatedcells are incubated in the light with provision of nitrogensource, but without glucose, they become green with active synthesisof chlorophyll and additional syntheses of RNA and protein. Based on these results and those to be reported later, it wasconcluded that the greening of the glucose-bleached cells involvesa light-independent phase followed by a light-requiring phasewhich entails the greening of cells and full organization ofchloroplasts, and that the latter process is essentially thesame as that taking place when the etiolated cells are incubatedin the light with provision of nitrogen source in the absenceof glucose. (Received September 5, 1964; )     

Studies on ribonucleic acids from Chlorella protothecoides with special reference to the degradation of chloroplast RNA during the process of "glucose-bleaching"

By growing Chlorella protothecoides under certain nutritionaland light conditions the following three different types ofalgal cells were obtained: (i) normal "green" cells grown ina medium rich in a nitrogen source (urea) and poor in glucoseunder illumination, (ii) "etiolated" cells cultivated in thesame medium in darkness, and (iii) "glucose-bleached" cellsgrown, in the light or in darkness, in a medium rich in glucoseand poor in the nitrogen source. The "glucose-bleached" cellscontain profoundly degenerated plastids, and the "etiolated"cells have only partially organized plastids. From these algalcells RNA was extracted by the cold phenol method, and fractionatedby MAK column chromatography and sucrose density gradient centrifugation,making use of ³²P-labelled E. coli RNA as the internal marker.It was found that in comparison with the green cells that arerich in chloroplast ribosomal RNA as well as in nonchloroplastic("cytoplasmic") ribosomal RNA, the etiolated cells possess acomparable amount of "cytoplasmic" rRNA but a significantlylesser amount of chloroplast rRNA. Both types of rRNA existat extremely low levels in the glucose-bleached cells. During the process of bleaching (chloroplast degeneration) ofthe green cells induced by the addition of a high concentrationof glucose, marked changes were observed in the patterns offractionation of RNA as followed by the above procedures. Itwas disclosed that the chloroplast rRNA is rapidly degradedduring an early phase of the bleaching process, while the quantityof "cytoplasmic" rRNA remained almost unaltered. ¹Part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965, and at the Symposium on Biogenesisof Subcellular Particles, the 7th Internatl. Congress of Biochemistry,Tokyo, 1967. ²Present address: Faculty of Pharmaceutical Sciences, Universityof Hokkaido, Sapporo.     

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; )     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides II. Role of carbamylphosphate in chloroplast regeneration

Levels of the activities of glutamine-dependent carbamylphosphatesynthetase, ornithine-and aspartate-transcabamylase and phosphoenolpyruvatecarboxylase were followed in greening cells of Chlorella prolothecoides.Among the enzymes examined the activity of carbamylphosphatesynthetase was extremely low, especially at the early phaseof greening. Arginine (but not ornithine or aspartate), when administeredto algal cells at the 24th hour of greening, stimulated thesyntheses of RNA, protein and chlorophyll in the subsequentperiod. It also affected the metabolic pathway of the ¹⁴CO₂supplied simultaneously with arginine in the presence of CMU.Arginine produced a decreased incorporation of ¹⁴C into proteinand an increased incorporation into nucleic acid. The mechanismof the action of CO₂ on chloroplast regeneration is discussed.We concluded that chloroplast regeneration in glucose-bleachedcells is limited by the synthesis of carbamylphosphate, especiallyin the early phase of greening. (Received August 19, 1975; )     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

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; )     

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; )     

Acetate metabolism in the process of "acetate-bleaching" of Chlorella protothecoides

Green cells of Chlorella protothecoides when incubated in amedium containing acetate but no nitrogen source, have beenshown to be bleached as strongly as in glucose-induced bleaching.Using U-¹⁴C-acetate as tracer, the acetate metabolism of algalcells during the process of acetate-induced bleaching was investigated.Changes in algal cell activities for respiration and assimilationof added ¹⁴C-acetate were followed during bleaching processesin "acetate-adapted" and "non-adapted" green cells. As in glucose-inducedbleaching of algal cells, algal cell activity for incorporating¹⁴C into lipids showed the most characteristic change, suggestingthat lipogenesis is causally related to the occurrence of bleachingin algal cells. (Received March 5, 1969; )     

SYNCHRONOUS CULTURE OF CHLORELLA: II. CHANGES IN CONTENT OF VARIOUS VITAMINS DURING THE COURSE OF THE ALGAL LIFE CYCLE

1. Chlorella ellipsoidea was grown synchronously and the changesin content of various vitamins during the algal life cycle werefollowed either by chemical or microbiological assay methods.
2. In terms of µg per gram of cell dry weight, the contentof some vitamins (niacin, biotin, inositol and choline) remainedalmost constant throughout the algal life cycle, while thatof others (vitamin B₆-complex, pantothenic acid, folic acid,thiamine and riboflavin) was found to decrease more or lessmarkedly during the "growing phase" and increase at later phasesof "ripening". The content of p-aminobenzoic acid increasedonly at an early stage of "ripening", and that of ascorbic acidincreased only at the stages in which photosynthesis occurredmost actively.
3. These results were discussed in an attemptto interprete theirrelationship with the previously reportedobservations pertainingto the physiological and biochemicalevents occurring in thelife cycle of the alga.

(Received November 7, 1959; )     

Factors Controlling Induction of External Carbonic Anhydrase and Change in K?(CO2) of Photosynthesis in Chlorella regularis

Transfer of algal cells of Chlorella regularis from 3% CO₂ inair into ordinary air in the light increased external carbonicanhydrase (CA) activity as well as photosynthetic affinity forCO₂ by several-fold within 2 h. Since no noticeable differencewas observed in CA activity between intact cells and cell homogenates,CA seemed to be mainly localized on the cell surface. Changesin CA activity and K_?(CO₂) of photosynthesis were not observedin the dark. CA induction was 50%-inhibited by incubation with10 µM DCMU during adaptation of high-CO₂ cells to air,whereas it was considerably suppressed when high-CO₂ cells preincubatedwith DCMU in the light for 6 h or without DCMU in the dark for24 h were used. The change in K_?(CO₂) of photosynthesis wasonly slightly affected by DCMU. Uncoupler like carbonylcyanide-m-chlorophenyl-hydrazone(CCCP) and inhibitors of mitochondrial respiration (KCN plussalicylhydroxamic acid) suppressed CA induction during adaptationof high-CO₂ cells to low CO₂ conditions. These results suggest that photosynthesis is not essential forCA induction in Chlorella regularis when some amounts of photosyntheticproducts are previously stored in the cells and respirationis active. A decrease in K_?(CO₂) of photosynthesis during adaptationfrom high to low CO₂ was mostly independent on photosynthesis.However, light is essential for both phenomena. (Received July 16, 1990; Accepted January 21, 1991)     

EFFECTS OF GLUCOSE ON THE PROCESS OF CHLOROPLAST DEVELOPMENT IN CHLORELLA PROTOTHECOIDES

By growing Chlorella protothecoides in a medium rich in glucoseand poor in nitrogen source (urea), the entirely chlorophyll-lesscells showing no discernible plastid structure and containingonly little RNA and protein were obtained. These cells, whichwere called "glucosebleached" cells, turned green after a certainlag period, when they were incubated, in the light, in a mediumcontaining the nitrogen source and basal mineral nutrients butwithout glucose. As has been shown in previous studies, thisgreening process involves two consecutive steps : a light-independentphase, in which RNA plays an essential role, and a light-requiringphase, in which the chlorophyll formation and full organizationof chloroplast take place accompanied by the formation of acertain species of protein ("alkali-stable" protein). The wholeprocess of greening was found to be profoundly suppressed byaddition of glucose, the degree of suppression being determinedby the relative concentrations of glucose and the nitrogen source.The primary act of glucose was manifest in the repression ofsyntheses of RNA, and of the species of protein mentioned above,which participate in, or causally related to, the first andsecond phases, respectively, in the greening process. ¹ This paper was read at the Symposium on Extranuclear Self-reproducingSystems held by the Botanical Society of Japan in October, 1964