The contrasting effects of chloramphenicol and cycloheximide on the recovery of cell division and chlorophyll synthesis in "giant" cells of Chlorella vulgaris (Emerson strain)

The simultaneous recovery of cell division and chlorophyll synthesisin "giant", "bleached" cells of the Emerson strain of Chlorellavulgaris which occurs upon exposure to light has been investigatedusing the two inhibitors of protein synthesis, chloramphenicoland cycloheximide. With both antibiotics, it has been foundpossible, under suitable conditions, to separate cell divisionand chlorophyll synthesis. The best separation is obtained withthose chloramphenicol treatments which severely inhibit chlorophyllsynthesis and the development of a photosynthetic capacity butwithout affecting cell division. The separation achieved withcycloheximide is less clear-cut. The significance of these resultsis discussed with particular reference to the relationship betweenchloroplast development and other events occurring in the cytoplasm. (Received October 12, 1970; )     

The role of photosynthesis and respiration in the light-induced recovery of "giant" cells of the Emerson strain of Chlorella

The light-induced recovery of cell division and chloroplastdevelopment in "giant", "bleached" cells of the Emerson strainof Chlorella is unaffected by treatments (atrazine. CMU, incubationin a CO₂-free atmosphere) which interfere with photosynthesis.Anaerobic conditions or the presence of respiratory inhibitors(DNP, KCN, NaN₃) markedly suppress recovery. Recovery is accompaniedby a mobilization of the reserve starch which follows a linearcourse over the first 9 hr. Chloramphenicol (50 µg/ml),which inhibits chlorophyll synthesis and the development ofa photosynthetic capacity, is without effect on the early rateof starch mobilization. Evidence is presented that the contributionof photosynthesis towards recovery is only significant whenthe reserve starch has been depleted. Recovery does not requirecontinuous light; the critical light-stimulated processes apparentlytaking place during the first 9 hr. The possible nature of thelight stimulation of recovery is discussed. (Received June 18, 1973; )     

Physiological changes accompanying the recovery of cell division in "giant" cells of Chlorella vulgaris (EMERSON strain)

A homogeneous population of "giant" cells of the EMERSON strainof Chlorella vulgaris, produced following culture under carefullycontrolled conditions in a glucose medium in the dark, recoversits capacity to undergo cell division when returned to autotrophicconditions. A similar recovery also occurs after a prolongedperiod of culture in the dark. The division of the giant cellsis accompanied, in each case, by marked pigment synthesis anda consequent recovery of photosynthetic capacity. Under autotrophicconditions the recovery of cell division and restoration ofthe full pigment concentration are complete within a 24 hr period.The recovery which takes place in a glucose medium in the darkoccurs only after a period of 10–14 days growth. (Received May 9, 1970; )     

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; )     

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; )     

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.     

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.     

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; )     

"GIGANTISM" OF CHLORELLA VULGARIS I. MECHANISM OF INDUCTION OF CIGANTISM

1. Based on the microscopic observations, two stages, "giant cellstage" and the subsequent "palmelloid body stage", were distinguishedin the process of formation of giant Chlorella induced by theaddition of sugars. The "giant cell" is much larger in sizethan the control cell, but the other morphological featuresare the same as those of the latter. The "palmelloid body" isa form composed of many conjoined autospores.
2. When a highconcentration of glucose was maintained in the medium,gigantismwas also maintained. Under this condition, the algashows acyclic transformation between "giant cell" and "palmelloidbody"without returning to the small single cells.
3. Large amountsof carbohydrate composed of hexose were foundto be accumulatedin the giant algal cells, and it was inferredthat this carbohydrateaccumulation causes greater enlargementof cell volume as comparedwith control cells.
4. Uronic acids, which were found to be absentin the control cells,were formed and lost in the cells culturedin the glucose mediumin parallel with the appearance and disappearanceof gigantism.
5. Pectic substances, from which uronic acids areconsidered tobe derived during the extraction procedure, werefound to bepresent only in giant Chlorella.
6. The conjoinedautospores in giant Chlorella (at the palmelloidbody stage)were separated to some extent by the addition ofEDTA, and theresulting cells were similar to control Chlorellacells.
7. Basedon these results it was inferred that inductive formationofthe pectic substances is causally related with the appearanceof "palmelloid body".

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

Effect of inhibitors of nucleic acid and protein synthesis on the reappearance of "light interruption rhythm" in a long-day duckweed, Lemna gibba G 3

Effects of several inhibitors of DNA, RNA and protein synthesison the reappearance of a once faded-out light interruption rhythmin a long-day duckweed, Lemna gibba G 3, were studied. The reappearancewas not affected by inhibitors of RNA and protein synthesis;i.e., 2-thiouracil, 8-azaguanine, ethionine and chloramphenicol,but was suppressed by inhibitors of DNA synthesis; i. e., 5-fluorodeoxyuridine,5-fluorouracil and mitomycin C only when these were appliedduring the light period for perturbation. We concluded that synthesis of a new DNA species during thelight period was required for the recurrence of this rhythm. (Received September 25, 1968; )     

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; )     

Division of Chloroplasts in a Green Alga, Trebouxia potteri

The division of chloroplasts in Trebouxia potteri was studiedby electron microscopy. At the beginning of chloroplast division,vesicles with fine fibrils (FVs) and ER attach to the isthmusof the chloroplast. Then, filaments appear around the isthmusparallel to the direction of constriction and seem to contractin order to decrease the diameter of the isthmus. It is suggestedthat the FVs are involved in the formation of the filamentsand that the ER is involved in the contraction of the filaments.At the final stages of the division of the chloroplast, thefilaments decompose. FVs are partially surrounded and decomposedby lysosomal sheets. For the next cycle of division of the chloroplast,the recovery of substances from decomposed filaments by functionalFVs seems a realistic possibility. chloroplast division, division apparatus, division cycle, transmission electron microscopy, Trebouxia potteri.     

Growth and Maintenance of an Embryogenic Cell Culture of Daylily {Hemerocallis) on Hormone-free Medium

The division of chloroplasts in Trebouxia potteri was studiedby electron microscopy. At the beginning of chloroplast division,vesicles with fine fibrils (FVs) and ER attach to the isthmusof the chloroplast. Then, filaments appear around the isthmusparallel to the direction of constriction and seem to contractin order to decrease the diameter of the isthmus. It is suggestedthat the FVs are involved in the formation of the filamentsand that the ER is involved in the contraction of the filaments.At the final stages of the division of the chloroplast, thefilaments decompose. FVs are partially surrounded and decomposedby lysosomal sheets. For the next cycle of division of the chloroplast,the recovery of substances from decomposed filaments by functionalFVs seems a realistic possibility. chloroplast division, division apparatus, division cycle, transmission electron microscopy, Trebouxia potteri     

Division of Chloroplasts in a Green Alga, Trebouxia potteri

The division of chloroplasts in Trebouxia potteri was studiedby electron microscopy. At the beginning of chloroplast division,vesicles with fine fibrils (FVs) and ER attach to the isthmusof the chloroplast. Then, filaments appear around the isthmusparallel to the direction of constriction and seem to contractin order to decrease the diameter of the isthmus. It is suggestedthat the FVs are involved in the formation of the filamentsand that the ER is involved in the contraction of the filaments.At the final stages of the division of the chloroplast, thefilaments decompose. FVs are partially surrounded and decomposedby lysosomal sheets. For the next cycle of division of the chloroplast,the recovery of substances from decomposed filaments by functionalFVs seems a realistic possibility. chloroplast division, division apparatus, division cycle, transmission electron microscopy, Trebouxia potteri     

RNA Synthesis in Phaseolus Chloroplasts: II. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLASTS FROM DEVELOPING AND SENESCING LEAVES

The rate of RNA synthesis in chloroplasts from the primary leavesof Phaseolus vulgaris L. cv. Canadian Wonder was measured invitro as plant age increased. The rate per leaf began to fallbefore the leaf was 70% expanded. At full expansion, activityhad fallen by 70%. Chloroplast RNA synthesis per unit chlorophyllwas falling before the leaf was 25% expanded. When all parts of the plant above the mature primary leaveswere removed (detopping) chloroplast RNA synthesis in theseleaves rose within 36 h. The rate increased to a maximum 3–4d after detopping, when it was 5–10 times control values;thereafter it fell again. The chlorophyll content began to increaseabout 4 d after detopping, eventually rising by 100%. Detoppingcaused a 3-fold increase in the Triton X-100-soluble DNA contentof chloroplast preparations, measured after 3.5 d. At that timethe rate of RNA synthesis per unit Triton-soluble DNA was thesame in chloroplasts from the primary leaves of intact and detoppedplants. Detopping also resulted in an increase in the depthof the leaf palisade layer. The effects of detopping on chloroplasts were prevented by darknessand reduced by shading. Increased chloroplast RNA polymerase activity was also inducedin the primary leaves by placing a polythene bag over intactplants, enclosing everything above these leaves. Removal ofthe roots from detopped plants prevented the rise in the rateof chloroplast RNA synthesis.     

Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division. In algal cells, chloroplast division is regulated by the cell cycle so as to occur only once, in the S phase. Chloroplasts possess multiple copies of their own genome that must be replicated during chloroplast proliferation. In order to examine how chloroplast DNA replication is regulated in the green alga Chlamydomonas reinhardtii, we first asked whether it is regulated by the cell cycle, as is the case for chloroplast division. Chloroplast DNA is replicated in the light and not the dark phase, independent of the cell cycle or the timing of chloroplast division in photoautotrophic culture. Inhibition of photosynthetic electron transfer blocked chloroplast DNA replication. However, chloroplast DNA was replicated when the cells were grown heterotrophically in the dark, raising the possibility that chloroplast DNA replication is coupled with the reducing power supplied by photosynthesis or the uptake of acetate. When dimethylthiourea, a reactive oxygen species scavenger, was added to the photoautotrophic culture, chloroplast DNA was replicated even in the dark. In contrast, when methylviologen, a reactive oxygen species inducer, was added, chloroplast DNA was not replicated in the light. Moreover, the chloroplast DNA replication activity in both the isolated chloroplasts and nucleoids was increased by dithiothreitol, while it was repressed by diamide, a specific thiol-oxidizing reagent. These results suggest that chloroplast DNA replication is regulated by the redox state that is sensed by the nucleoids and that the disulfide bonds in nucleoid-associated proteins are involved in this regulatory activity.Chloroplasts are semiautonomous organelles that possess their own genome, which is complexed with proteins to form nucleoids and also certain machinery needed for protein synthesis, as is the case in prokaryotes. It is generally accepted that chloroplasts arose from a bacterial endosymbiont closely related to the currently extant cyanobacteria (Archibald, 2009; Keeling, 2010). In a manner reminiscent of their free-living ancestor, chloroplasts proliferate by the division of preexisting organelles that are coupled to the duplication and segregation of the nucleoids (Kuroiwa, 1991) and have retained the bulk of their bacterial biochemistry. However, chloroplasts have subsequently been substantially remodeled by the host cell so as to function as complementary organelles within the eukaryotic host cell (Rodríguez-Ezpeleta and Philippe, 2006; Archibald, 2009; Keeling, 2010). For example, most of the genes that were once in the original endosymbiont genome have been either lost or transferred into the host nuclear genome. As a result, the size of the chloroplast genome has been reduced to less than one-tenth that of the free-living cyanobacterial genome. Thus, the bulk of the chloroplast proteome consists of nucleus-encoded proteins that are translated on cytoplasmic ribosomes and translocated into chloroplasts. In addition, chloroplast division ultimately came to be a process tightly regulated by the host cell, which ensured permanent inheritance of the chloroplasts during the course of cell division and from generation to generation (Rodríguez-Ezpeleta and Philippe, 2006; Archibald, 2009; Keeling, 2010).Chloroplast division is performed by constriction of the ring structures at the division site, encompassing both the inside and the outside of the two envelopes (Yang et al., 2008; Maple and Møller, 2010; Miyagishima, 2011; Pyke, 2013). One part of the division machinery is derived from the cyanobacterial cytokinetic machinery that is based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including one member of the dynamin family. The majority of algae (both unicellular and multicellular), which diverged early within the Plantae, have just one or at most only a few chloroplasts per cell. In algae, the chloroplast divides once per cell cycle before the host cell completes cytokinesis (Suzuki et al., 1994; Miyagishima et al., 2012). In contrast, land plants and certain algal species contain dozens of chloroplasts per cell that divide nonsynchronously, even within the same cell (Boffey and Lloyd, 1988). Because land plants evolved from algae, there is likely to have been a linkage between the cell cycle and chloroplast division in their algal ancestor that was subsequently lost during land plant evolution. Our recent study showed that the timing of chloroplast division in algae is restricted to the S phase by S phase-specific formation of the chloroplast division machinery, which is based on the cell cycle-regulated expression of the components of the chloroplast division machinery (Miyagishima et al., 2012).Because chloroplasts possess their own genome, chloroplast DNA must be duplicated so that each daughter chloroplast inherits the required DNA after division. However, it is still unclear how the replication of chloroplast DNA is regulated and whether the replication is coupled with the timing of chloroplast division, even though certain studies have addressed this issue, as described below.Bacteria such as Escherichia coli and Bacillus subtilis possess a single circular chromosome. In these bacteria, the process of DNA replication is tightly coupled with cell division (Boye et al., 2000; Zakrzewska-Czerwińska et al., 2007), in which the initiation of replication is regulated such that it occurs only once per cell division cycle (Boye et al., 2000). In contrast, cyanobacteria contain multiple copies of their DNA (e.g. three to five copies in Synechococcus elongatus PCC 7942; Mann and Carr, 1974; Griese et al., 2011). In some obligate photoautotrophic cyanobacterial species, replication is initiated only when light is available (Binder and Chisholm, 1990; Mori et al., 1996; Watanabe et al., 2012). Replication is initiated asynchronously among the multiple copies of the DNA. Although the regulation of the initiation of DNA replication is less stringent than that in E. coli and B. subtilis, as described above, a recent study using S. elongatus PCC 7942 showed that this replication peaks prior to cell division, as in other bacteria.Chloroplasts also contain multiple copies of DNA (approximately 1,000 copies; Boffey and Leech, 1982; Miyamura et al., 1986; Baumgartner et al., 1989; Oldenburg and Bendich, 2004; Oldenburg et al., 2006; Shaver et al., 2008). In algae, chloroplast DNA is replicated in a manner that keeps pace with chloroplast and cell division in order to maintain the proper DNA content per chloroplast (i.e. per cell). In contrast, in land plants, the copy number of DNA in each chloroplast (plastid) changes during the course of development and differentiation, although contradictory results were reported about leaf development (Lamppa and Bendich, 1979; Boffey and Leech, 1982; Hashimoto and Possingham, 1989; Kuroiwa, 1991; Rowan and Bendich, 2009; Matsushima et al., 2011). Previous studies that synchronized the algal cell cycle by means of a 24-h light/dark cycle showed that chloroplast DNA is replicated only during the G1 phase, after which it is separated into daughter chloroplasts during the S phase by chloroplast division, implying that chloroplast DNA replication and division are temporally separated (Chiang and Sueoka, 1967; Grant et al., 1978; Suzuki et al., 1994). However, under these experimental conditions, G1 cells grow and the chloroplast DNA level increases during the light period. Cells enter into the S phase, chloroplast DNA replication ceases, and the chloroplasts divide at the beginning of the dark period. Thus, it is still unclear whether chloroplast DNA replication is directly controlled by the cell cycle, as is the case in chloroplast division, or chloroplast DNA replication occurs merely when light energy is available.We addressed this issue using a synchronous culture as well as a heterotrophic culture of the mixotrophic green alga Chlamydomonas reinhardtii. The results show that chloroplast DNA replication occurs independently of either the cell cycle or the timing of chloroplast division. Instead, it is shown that chloroplast DNA replication occurs when light is available in photoautotrophic culture and even under darkness in heterotrophic culture. Further experimental results suggest that chloroplast DNA replication is regulated by the redox state in the cell, which is sensed by the chloroplast nucleoids.     

The Chloroplast and Leaf Developmental Mutant, pale cress, Exhibits Light-conditional Severity and Symptoms Characteristic of its ABA Deficiency

The PALE CRESS gene (PAC) is essential for proper chloroplastand leaf development in Arabidopsis thaliana. The ability ofpac mutants to accumulate significantly more chlorophyll whengrown in low light conditions than in high light conditionssuggests that carotenoid deficiency is at least partly responsiblefor premature cessation of chloroplast development. In additionto accumulation of low levels of chlorophyll and carotenoidpigments,pac mutants are abscisic acid (ABA) deficient and havecharacteristics which may be explained by this deficiency. Theseinclude reduced seed viability and, in enclosed growth conditions,increased leaf growth. Plants transformed with an antisensePAC construct often bear viviparous embryos which may be symptomaticof a deficiency in ABA. Since carotenoids are precursors ofABA, a role for PAC in carotenoid biosynthesis is further supported.The nuclear-encoded, chloroplast-localized PAC protein has beenimplicated in the maturation of plastid-encoded mRNAs. Thus,PAC may affect the abundance of one or more chloroplast proteinswhich function in the synthesis or stability of carotenoids.Using thePROLIFERA gene as a marker for cell division, it isshown that cell division profiles in the pac shoot apex aredisrupted. pac leaves are relatively normal in size and shapedespite the light intensity-induced variability of leaf celldefects. Copyright 2000 Annals of Botany Company Abscisic acid, carotenoid, chloroplast development, leaf development, organismal theory, PALE CRESS,PROLIFERA , vivipary     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

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; )