Effect of cycloheximide on the process of "glucose-bleaching" in Chlorella protothecoides

The process of bleaching of Chlorella protothecoides inducedby the addition of glucose was strongly inhibited by cycloheximide,an inhibitor of protein synthesis, whereas it was suppressedonly weakly by chloramphenicol, puromycin and ethionine. Whencycloheximide was added simultaneously with glucose at the beginningof die bleaching experiment, no bleaching of algal cells occurredduring the subsequent incubation. When it was added after glucose,the bleaching of algal cells proceeded for a period of timeas actively as in the control, then gradually ceased. Cycloheximidewas found to suppress the uptake of glucose by algal cells,and to severely inhibit the assimilation of glucose into lipidswhen added at the beginning of the bleaching experiment. Theseinhibitory effects of cycloheximide are discussed in relationto the induction of "glucose-bleaching" in algal cells. (Received December 16, 1968; )     

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; )     

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; )     

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; )     

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; )     

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; )     

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; )