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; )     

NUTRITIONAL CONTROL OF CELL PIGMENTATION IN CHLORELLA PROTOTHECOIDES WITH SPECIAL REFERENCE TO THE DEGENERATION OF CHLOROPLAST INDUCED BY GLUCOSE

1. Measuring the chlorophyll contents and growth of the algalcells grown in media containing different amounts of glucose(G) and urea fas a nitrogen source, N), it was found that theratio N/G determines pigmentation of the cells (colourless,yellow, yellowish green and green) under the experimental conditionsused, and thus a sort of colour map of the differently pigmentedcells was obtained. 2. The bleached cells produced at lower N/G ratios and the greencells obtained at higher ratios could he cultured successivelyunder heterotrophic and photoautotrophic conditions, respectively,and both forms were interconvertible on transferring each celltype into a new medium having appropriate N/G ratio. 3. Studies on these bleaching and greening processes under differentexperimental conditions revealed that the greening requiresessentially the supply of N-sources—no strict specificitywas observed with different N-sources tested—as well aslight, but can take place independently of growth, while thebleaching is caused most strongly by glucose (and fructose)among the carbon sources examined and proceeds essentially independentof light. 4. The bleaching effect of glucose at its higher concentrationsis primarily due to its degradation effect on chloroplast structuresincluding lamellae. This effect of glucose is markedly diminishedat its decreasing concentrations and is also counteracted bythe supply of N-source (urea) of higher concentrations. ¹ This work was partly reported at the Symposium on Cell Differentiationsponsored by the Society of Agricultural Chemistry, Japan, inApril, 1963 and at the Symposium on Nitrogen and Plant by theJapanese Society of Plant Physiologists in October, 1963.     

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; )     

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.     

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; )     

THE ROLE OF RESPIRATION AND PHOTOSYNTHESIS IN THE CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, entirely chlorophyll-less cellsof Chlorella protothecoides are obtained when the alga is grownin a medium rich in glucose and poor in nitrogen source (urea).These cells, which are referred to as "glucose-bleached" cells,have neither discernible chloroplast structures nor photosyntheticactivity. When the "glucose-bleached" cells are incubated, inthe light, in a nitrogen-enriched mineral medium without addedglucose, they turn green, after an induction period, with regenerationof chloroplasts and development of the capacity for performingnormal photosynthesis. In the present study, changes in respiratoryactivity of algal cells during the process of greening (chloroplastregeneration) were followed, and the effects of various inhibitorsof respiration and photosynthesis on the greening process wereexamined. 2. The glucose-bleached cells showed a very low activity ofrespiration, and the activity increased markedly during an earlyphase of chloroplast regeneration, showing, however, a decreaseduring the subsequent phase of greening. 3. Some antimetabolites which inhibited the cell respiration,were found to suppress also the greening of cells. 2,4-Dinitrophenoland azide, potent inhibitors of oxidative phosphorylation, acceleratedconsiderably both the respiration and greening of algal cells.CMU inhibited completely photosynthesis of the greening cells,but suppressed only slightly the greening process. 4. Based on these results it was concluded that the primaryrole of respiration in the chloroplast regeneration in the glucose-bleachedcells is to produce oxidized carbon compounds (and perhaps reducedforms of NAD and NADP) for various biosynthetic reactions. Itwas further suggested that ATP may be supplied for the chloroplastregeneration by a certain means different from the oxidativephosphorylation or photophosphorylation. The activities of photosyntheticphosphorylation and CO₂-fixation developing in the greeningcells do not appear to play any essential role in the chloroplastregeneration. (Received December 27, 1965; )     

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; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: V. DEGENERATION OF CHLOROPLASTS INDUCED BY DIFFERENT CARBON SOURCES, AND EFFECTS OF SOME ANTIMETABOLITES UPON THE PROCESS INDUCED BY GLUCOSE

1. The green cells of Chlorella protothecoides were bleached todifferent extents when incubated (in the dark) in the nitrogen-freemedia containing, besides basal mineral nutrients, glucose,fructose, galactose, glycerol or acetate. Glucose and fructosewere found to have the strongest bleaching effect. Additionof a nitrogen source (urea) caused a considerable reductionof the bleaching. It was assumed that from the different carbonsources a certain common intermediate(s) causing the bleachingis formed, and that in the presence of the nitrogen source thesubstance is removed by reacting with it.
2. Using glucose asbleach-inducing agent, the effects of someantimetabolites uponthe processes of bleaching, division andgrowth of green algalcells were investigated, and it was demonstratedthat the processof bleaching occurs without being accompaniedby growth anddivision of the algal cells.
3. It was found that during theprocess of bleaching no net increasesin RNA and protein tookplace.

(Received March 11, 1965; )     

FURTHER NOTES ON THE GROWTH AND CHLOROPHYLL FORMATION OF CHLORELLA PROTOTHECOIDES

1. Using area of a fixed concentration as a nitrogen source,cells of Chlorella protothecoides were grown in the presenceof various carbon compounds. Magnitudes of growth of the cellswere widely different depending on the carbon sources used;glucose and fructose being most favourable substrates and galactose,glycerol and acetate coming next. But the amounts of chlorophyllformed in the cells during the experimental period were almost,the same irrespective of the different carbon sources, withsome exceptions. The similarity of the chlorophyll level observedin these experiments seemed to indicate that the formation ofchlorophyll was limited largely by the nitrogen source but notby the carbon source. 2. Strong bleaching effect was recognized with glucose and fructoseat their high concentration, which produced totally chlorophyll-lesscells. On the other hand, a stimulating effect on chlorophyllformation was observed with galactose at the different concentrationsexamined. 3. Effects of glycine and ammonium carbonate as the nitrogensource on the algal growth and pigmentation were studied insome details. The results were similar to those previously obtainedwith urea, confirming our previous conclusion that the algalpigmentation is profoundly affected by the concentration balancebetween glucose and nitrogen source. ¹Present address: Tokyo Research Laboratories, Tanabe SeiyakuCo., Toda-machi, Saitama.     

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; )     

THE EFFECTS OF VARIATIONS IN THE CONCENTRATION OF OXYGEN AND OF GLUCOSE ON DARK ADAPTATION

In this study we have analyzed the effects of variations in the concentrations of oxygen and of blood sugar on light sensitivity; i.e. dark adaptation. The experiments were carried out in an air-conditioned light-proof chamber where the concentrations of oxygen could be changed by dilution with nitrogen or by inhaling oxygen from a cylinder. The blood sugar was lowered by the injection of insulin and raised by the ingestion of glucose. The dark adaptation curves were plotted from data secured with an apparatus built according to specifications outlined by Hecht and Shlaer. During each experiment, observations were first made in normal air with the subject under basal conditions followed by one, and in most instances two, periods under the desired experimental conditions involving either anoxia or hyper- or hypoglycemia or variations in both the oxygen tension and blood sugar at the same time. 1. Dark adaptation curves were plotted (threshold against time) in normal air and compared with those obtained while inhaling lowered concentrations of oxygen. A decrease in sensitivity was observed with lowered oxygen tensions. Both the rod and cone portions of the curves were influenced in a similar way. These effects were counteracted by inhaling oxygen, the final rod thresholds returning to about the level of the normal base line in air or even below it within 2 to 3 minutes. The impairment was greatest for those with a poorer tolerance for low O₂. Both the inter- and intra-individual variability in thresholds increased significantly at the highest altitude. 2. In a second series of tests control curves were obtained in normal air. Then while each subject remained dark adapted, the concentrations of oxygen were gradually decreased. The regeneration of visual purple was apparently complete during the 40 minutes of dark adaptation, yet in each case the thresholds continued to rise in direct proportion to the degree of anoxia. The inhalation of oxygen from a cylinder quickly counteracted the effects for the thresholds returned to the original control level within 2 to 3 minutes. 3. In experiments where the blood sugar was raised by the ingestion of glucose in normal air, no significant changes in the thresholds were observed except when the blood sugar was rapidly falling toward the end of the glucose tolerance tests. However, when glucose was ingested at the end of an experiment in low oxygen, while the subject remained dark adapted, the effects of the anoxia were largely counteracted within 6 to 8 minutes. 4. The influence of low blood sugar on light sensitivity was then studied by injecting insulin. The thresholds were raised as soon as the effects of the insulin produced a fall in the blood sugar. When the subjects inhaled oxygen the thresholds were lowered. Then when the oxygen was withdrawn so that the subject was breathing normal air, the thresholds rose again within 1 to 2 minutes. Finally, if the blood sugar was raised by ingesting glucose, the average threshold fell to the original control level or even below it. 5. The combined effects of low oxygen and low blood sugar on light sensitivity were studied in one subject (W. F.). These effects appeared to be greater than when a similar degree of anoxia or hypoglycemia was brought about separately. 6. In a series of experiments on ten subjects the dark adaptation curves were obtained both in the basal state and after a normal breakfast. In nine of the ten subjects, the food increased the sensitivity of the subjects to light. 7. The experiments reported above lend support to the hypothesis that both anoxia and hypoglycemia produce their effects on light sensitivity in essentially the same way; namely, by slowing the oxidative processes. Consequently the effects of anoxia may be ameliorated by giving glucose and the effects of hypoglycemia by inhaling oxygen. In our opinion, the changes may be attributed directly to the effects on the nervous tissue of the visual mechanism and the brain rather than on the photochemical processes of the retina.     

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: 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; )     

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.     

一个新的水稻斑马叶突变(zebraleaf1)对叶绿体发育的影响(简报)

通过γ射线诱变,在水稻粳稻栽培品种9522中得到一个斑马叶突变体zebra leaf 1.为了研究zl1的功能,我们对突变体进行了形态学和细胞学的分析,同时也对此基因突变以后对叶绿体发育和光合作用的影响作了评价.突变体叶片上绿色和枯白色条纹相同,叶绿素含量显著的下降.电镜显示叶绿体类囊体的排列被打乱,变得杂乱无章.这表明zl1突变体在叶绿体发育过程中出现障碍.zl1基因的突变使得净光合速率显著的下降.参与光合作用的一些关键蛋白,比如核酮糖1,5-二磷酸羧化酶/加氧酶(Rubisco)、Rubisco活化酶、Dl蛋白、CF1β亚基的表达量也显著的下调.但是,zl1突变体对外界环境非常敏感,有时会没有表型.