Photosynthetic Activity during the Division Cycle in Synchronized Euglena gracilis

Axenic populations of the photosynthetic protozoan Euglena gracilis, grown with autotrophic nutrition, were synchronized with respect to cell division by culture on an alternating light-dark cycle. No cell divisions occurred in the light periods; approximately 100% of the cells divided in the dark periods. In such cultures, the synthesis of photosynthetic pigments and accumulation of polysaccharide were confined to the light periods. The capacity for photosynthesis, however, increased continuously over the entire light-dark cycle, and is thus not directly correlated with pigment content. A correlation was seen between photosynthetic capacity and protein content, suggesting that enzymatic mechanisms of the photosynthetic apparatus might be the limiting factor. Estimates of total photosynthetic activity indicate that about 5 x 10(-6) calories are required for the synthesis of a new cell.     

The s phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G1 by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

The S phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G₁ by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

Synchronous growth and synthesis of macromolecules in a naturally wall-less volvocale,Dunaliella bioculata

Summary Dunaliella bioculata, a naturally wall-less unicellular green alga, can be induced to divide synchronously when subjected to a 12 hours light-12 hours dark cycle. This rhythmic cell division will last for at least 15 days under a subsequent constant illumination. Synchronization can be improved when cells are submitted to 8 hours light-16 hours dark cycles under bright white light (10,000 lux). In these conditions the cell division gives rise to two daughter cells: The chronology of DNA, RNA and proteins synthesis has been studied during such a synchronized cell cycle. DNA synthesis begins 4 hours before the outset of cell division and is completed after two hours in the dark; in difference, illumination seems necessary to the synthesis of RNA and proteins.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary Cell division in Navicula pelliculosa (Bréb.) Hilse, strain 668 was synchronized with an alternating regime of 5 h light and 7 h dark. Cell volume and dry weight increased only during the light period. DNA synthesis, which began during the third h of light, was followed sequentially by mitosis, cytokinesis, silicic acid uptake, cell wall formation, and cell separation. Silicification and a small amount of net synthesis of DNA, RNA and protein occurred during the dark at the expense of carbohydrate reserves accumulated during the light period. Cells kept in continuous light, after synchronization with the light-dark regime, remained synchronized through a second division cycle; the sequence of morphological events was the same as that in the light-dark division cycle, but the biosynthesis of macromolecular components changed from a stepwise to a linear pattern. The silicon-starvation synchrony was improved by depriving light-dark synchronized cells of silicic acid at the beginning of their division cycle, then resupplying silicic acid to cells blocked at wall formation.Abbreviation L light - D dark Portions based on a thesis submitted by W.M.D. to the University of California, San Diego in partial fulfillment of the requirements for the PH.D degree     

Interactions between the nucleus and cytoplasmic organelles during the cell cycle of Euglena gracilis in synchronized cultures. IV. An aggregate form of chloroplasts in association with the nucleus appearing prior to chloroplast division

Cells of Euglena gracilis were synchronized by applying a 14-h light:10-h dark regimen under photoautotrophic conditions and a 10-h light:14-h dark regimen under photoorganotrophic conditions. At a stage just prior to chloroplast division in the cell cycle of these synchronized cultures, chloroplasts temporarily gathered in the posterior part of the cell and were connected to each other by many bridges. Part of the chloroplast aggregate surrounded about half of the nuclear surface, making connections or close contacts at many sites. A chromosome was always attached to the inner membrane of the nuclear envelope at the site of association with the chloroplast. The nucleoids in these aggregate chloroplasts, examined by staining with 4',6-diamidino-2-phenylindole, a DNA fluorochrome, showed profiles of strings or strands with branchings, under photoorganotrophic conditions at least, and some parts of the branchings came close to the site of association with the nucleus. The association between the chloroplast aggregate and the nucleus was also observed in Euglena cells placed in continuous darkness after synchronization under photoorganotrophic conditions, suggesting that these organellar associations are related to the Euglena cell cycle but are not the result of light:dark alternations used for cell synchronization.     

Dark incubation causes reinitiation of cell cycle events in Anacystis nidulans.

Synchronized cultures of Anacystis nidulans (Synechococcus PCC 6301), an obligate phototroph, are obtained by incubating exponential cultures in the dark for 12 to 16 h. A temporal and sequential order of macromolecular synthesis is observed within the cell division cycle of a synchronously dividing culture in the light. Apparently, dark incubation causes the cells to realign their cellular activities in such a way that all cells emerge from the dark and grow synchronously in the light. A study was conducted to explore the possible mechanisms responsible for the putative dark-induction process. Samples were taken at various times from a synchronized culture and were subjected to another round of dark incubation for 16 h. When these cultures were returned to the light, the cell number increased from 3 h and doubled at about 7 h. The protein, RNA, and DNA contents started to increase in order well before 3 h. This general pattern of cellular activities, observed for nearly all samples (i.e., for cells of different physiological ages), indicated that the dark incubation period caused the ongoing cell cycle to abort and a new cell cycle to be reinitiated under light growth conditions.     

Cyclic Changes in Chloroplast Structure in Synchronized Euglena gracilis*

SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.     

SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

Inductive and Inhibitory Effects of Light on Cell Division in Chattonella antiqua

The cell division of a red tide flagellate, Chattonella antiqua,was synchronously induced under light and dark regimes of 10L14D(a light period, L, for 10 h followed by a dark period, D, for14 h), 12L12D and l4L10D. In all regimes cell number began toincrease ca. 14 h after the onset of L and almost doubled duringone LD cycle. When the light-off timing of the last L was changedor the whole L was shifted, cells that had been synchronizedunder 12L12D invariably began to divide ca. 14 h after the onsetof L. This shows that the timing of cell division was determinedby the time of the onset of L. When cells were continuously exposed to light after a cell division,the subsequent cell division was inhibited. This effect waslimited to cells that had been synchronized under short-dayconditions. Thus it can be concluded that light has both inductive and inhibitoryeffects on cell division in this alga, the latter effect dependingupon the previously given light and dark regimes. (Received December 21, 1984; Accepted February 28, 1985)     

Change in Photosynthetic Capacity over the Cell Cycle in Light/Dark-Synchronized Amphidinium carteri Is Due Solely to the Photocycle

Cell cycle dependent photosynthesis in the marine dinoflagellate Amphidinium carteri was studied under constant illumination and light/dark (L/D) photocycles to distinguish intrinsic cell cycle control from environmental influences. Cells were grown in constant light and on a 14:10 L:D cycle at light intensities that would yield a population growth rate of 1 doubling per day. In the former case division was asynchronous, and cells were separated according to cell cycle stage using centrifugal elutriation. Cells grown on the L:D cycle were synchronized, with division restricted to the dark period. Cell cycle stage distributions were quantified by flow cytometry. Various cell age groups from the two populations were compared as to their photosynthetic response (photosynthetic rate versus irradiance) to determine whether or not the response was modulated primarily by cell cycle constraints or the periodic L/D cycle. Cell cycle variation in photosynthetic capacity was found to be determined solely by the L/D cycle; it was not present in cells grown in constant light.     

A possible second role for calmodulin in biological clock-controlled processes of euglena

The response of the Euglena gracilis (Klebs strain Z) photosynthesis circadian rhythm to three calmodulin antagonists was examined. In the presence of an antagonist, the photosynthetic reactions were uncoupled from the biological clock. Instead of the highly predictable rhythmic pattern characteristic of a biological clock-controlled circadian rhythm, the photosynthetic rate appears to be influenced by the light/dark cycle. The rate of O₂ evolution increases throughout the light portion of the cycle and does not decrease until the cells are exposed to darkness. Shortterm exposure to a calmodulin antagonist (2 hour pulses) failed to cause phase shifts in the timing of the rhythm. This suggests that calmodulin is not part of the clock controlling photosynthesis and that it has a clock-related role different from that reported for the cell division rhythm in Euglena.     

Stage-dependent localization of LHCP II apoprotein in the Golgi of synchronized cells of Euglena gracilis by immunogold electron microscopy

We have localized LHCP II apoprotein in the Golgi and thylakoids of Euglena gracilis Klebs var. bacillaris Cori and strain Z Pringsheim by electron microscopy using a specific antibody and protein A-gold. Using synchronized cells (light, 14 h:dark, 10 h) we show that thylakoids are always immunoreactive. There is no reaction in the Golgi at 0 h (the beginning of the light period) but immunoreaction appears in the Golgi soon thereafter, rises to a peak at 8 h and declines to zero by 16 h (2 h into the dark period). The peak in immunoreaction in the Golgi immediately precedes the peak in cellular 14C-labeling of thylakoid LHCP II apoprotein seen by Brandt and von Kessel (Plant Physiol. (1983) 72, 616), supporting our suggestion that processing in the Golgi precedes deposition of LHCP apoprotein in the thylakoids. Substitution of preimmune serum for antiserum eliminates the immunoreaction in the Golgi, and thylakoids of synchronized cells of mutant Gr1BSL which lacks LHCP II apoprotein show no immunoreaction in the Golgi or thylakoids at any stage. Random observations indicate that the compartmentalized osmiophilic structure (COS) shows an immunoreaction with anti-LHCP II apoprotein antibody at 1 h into the light period (when the Golgi is not immunoreactive) and at 10 h into the light period (when the Golgi is fully reactive), suggesting that the COS remains immunoreactive throughout the cell cycle.     

Periodicity in cell division and physiological behavior of ditylum brightwellii,a marine planktonic diatom,during growth in light-dark cycles

Summary Cells of Ditylum brightwellii, a large marine centric diatom, were partially synchronized by employing an appropriate light-dark cycle. At 20°C this consisted of 8 hrs of illumination at an intensity of 0.05 cal/cm² min. A single 2.8 l culture was studied over a 20 day period by diluting the culture daily to a standard cell concentration. The sequence of events in cell development was as follows: daughter cells were formed late in the light period, in the dark they elongated and the numerous chromatophores began dividing. A minimum cell buoyancy was observed in the dark concurrent with cell elongation. Increase in cell phosphorus took place in the dark period. The photosynthetic rate of cells removed during the dark period decreased to a minimum. In the following light period photosynthetic rate increased to a maximum, photosynthetic pigments, cell carbon, nitrogen, and carbohydrate increased and cell division again took place. Cell silica content increased concomitant with cell division. Details of cell morphology during cell division, based upon light microscopy, are reported.Contribution of the Scripps Institution of Oceanography.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Sequential changes in cell volume distribution during vitamin B12 starvation of Euglena gracilis.

During vitamin B12 starvation of Euglena, a new peak appears in the cell volume distribution. Some cells are inhibited at a unique point in the cell cycle between the initiation of DNA synthesis and nuclear division. The mechanism of inhibition of other cells differs.     

Evolution of ornithine decarboxylase activity during the cell cycle of Euglena gracilis Z in synchronous culture. Influence of vitamin B-12.

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark. During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G1 phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G2. In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.     

Perspectives in the coordinate regulation of cell cycle events in Synechococcus

The concepts of cell theory and the notions of coordinate regulation of the cell cycle have been known for centuries but the conundrum of coordinate regulation of the cell cycle remains to be resolved. The unique characteristics of the cell division cycle of Synechococcus, a photosynthetic bacterium, suggest the existence of a complex network of light/dark responsive gene regulatory factors that coordinate its cell cycle events. Evaluation of the highly ordered cell cycle of Synechococcus led to the construction of workable models that coordinate the cell cycle events. A central issue in bacterial cell growth is the elucidation of the genetic regulatory mechanisms that coordinate the cell cycle events. Synechococcus, a unicellular cyanobacterium, displays a peculiar cell growth cycle. In the light growth conditions, a highly ordered and sequentially coordinated appearances of r-protein synthesis, rRNA synthesis, DNA replication, chromosome segregation, and cell septum formation occur (Figs 1, 2A). Cell membrane syntheses occur predominantly during mid-cell cycle and cell division period. Synthesis of thylakoid (=photosynthetic apparatus) is thought to occur during mid-cell cycle and coincides with a period of peak phospholipid synthesis and oxygen production (Csatorday and Horvath, 1977; Asato, 1979). Cell wall syntheses occur in short discontinuous periods throughout the cell cycle and during cell division (Asato, 1984). Distinct D1 (=G1), C (S) and D2 (=G2) periods as defined by Cooper and Helmstetter (1968) are observed in synchronized cultures of Synechococcus (Asato, 1979). When light grown cultures are placed in the dark, the ongoing cell cycles are aborted in the dark (Fig. 3A) and cell divisions do not occur (Asato, 1983; Marino and Asato, 1986). Upon re-exposure of the cell cultures to the light growth conditions, about 14 h later, new cell cycles are re-initiated. These characteristics of cell growth are considered to be expressions of a unique strategy of obligate phototrophic mode of growth to perpetuate their species (Asato, 2003). Nevertheless, the intermediate metabolism, the synthesis of building block molecules, the genetics and molecular biology in the formation of major macromolecules are similarto heterotrophs such as E. coli. In any case, the genes that are involved in the formation of the cellular structures and the genes that control the orderly appearances of the cell cycle events must be coordinated by novel genetic mechanisms. Currently, there are no known physiological/physical mechanisms, growth rate dependent factors or traditional genetic regulatory mechanisms that could explain the coordinate regulation of the cell cycle events in bacteria (Newton and Ohta, 1992; Vinella and D'Ari, 1995; Donachie, 2001; Margolin and Bernander, 2004). Because the genetic mechanisms of coordinate regulation of cell cycle events in bacteria are largely unexplained, the questions on how Synechococcus coordinates the cell cycle events present a difficult problem to resolve. Nevertheless, the problems with regard to the coordinate regulation of the cell cycle events of Synechococcus must be considered. Possible solutions are developed and described in this article. The proposed schemes do not exclude the formation of other genetic mechanisms on the regulation of cell cycle events in Synechococcus. Although the cell cycle of Synechococcus is not widely known, the issues on the coordinate regulation of the cell cycle events are not trivial since similar regulatory mechanisms most likely occur in other prokaryotes.