Blue light delays commitment to cell division in Chlamydomonas reinhardtii

In this study, we describe the effect of red and blue light on the timing of commitment to cell division in Chlamydomonas reinhardtii. The time point and cell size after which cells can complete their cell cycle with one division round were determined for cultures that were exposed to various red and blue light periods. We show that the commitment point of cells grown in blue light is shifted to a later time point and a larger cell size, when compared with cells grown in red light. This shift was reduced when cultures were exposed to shorter blue light periods. Furthermore, this shift occurred only when exposure to blue light started before the cells attained a particular size. We conclude that the critical cell size for cell division, which is the cell size at which commitment to cell division is attained, is dependent on spectral composition.     

Phytochrome action on the timing of cell division in adiantum gametophytes

When filamentous protonemata of Adiantum capillus-veneris L. precultured under continuous red light were transferred to the dark, the apical cell divided about 24 to 36 hours thereafter. The time of the cell division was delayed for several hours by a brief exposure to far red light given before the dark incubation. The effect of far red light was reversed by a small dose of red light given immediately after the preceding far red light. The effects of red and far red light were repeatedly reversible, indicating that the timing of cell division was regulated by a phytochrome system. When a brief irradiation with blue light was given before the dark incubation, the cell division occurred after 17 to 26 hours in darkness. A similar red far red reversible effect was also observed in the timing of the blue light-induced cell division. Thus, the timing of cell division appeared to be controlled by phytochrome and a blue light-absorbing pigment.     

Division of chloroplast nucleoids and replication of chloroplast DNA during the cell cycle ofDunaliella salina grown under blue and red light

Summary Synchronous cultures of the algaDunaliella salina were grown in blue or red light. The relationships between replication of chloroplast DNA, cell size, cell age and the number of chloroplast nucleoids were studied. The replication of chloroplast DNA and the division of chloroplast nucleoids occurred in two separate periods of the chloroplast cycle. DNA replication was concomitant with that in the nucleocytoplasmic compartment but nucleoid division occurred several hours earlier than nuclear division. Red-light-grown cells were bigger and grew more rapidly than those grown in blue light. In newly formed daughter cells, the chloroplast nucleoids were small and spherical and they were localized around the pyrenoid. During the cell cycle they spread to other parts of the chloroplast. The number of DNA molecules per nucleoid doubled during DNA replication in the first third of the cell cycle but decreased several hours later when the nucleoids divided. Their number was fairly constant independent of the different light quality. Cells grown in red light replicated their chl-DNA and divided their nucleoids before those grown in blue light and their daughter cells possessed about 25 nucleoids as opposed to 15.Abbreviations DAPI 4,6-diamidino-2-phenylindole - chl-DNA chloroplast DNA - PAR photosynthetically active radiation     

The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal algaScenedesmus obliquus

The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures of Scenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100 % of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess approximately 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed.     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Effects of narrow-beam irradiations with blue and far-red light on the timing of cell division in Adiantum gametophytes

Protonemata of the fern Adiantum capillusveneris L., grown as single-cell filaments under continuous red light, were irradiated with a narrow beam of blue light. Only irradiation of the region containing the nucleus induced cell division. Beams of 30 m in width, which corresponds to the diameter of the nucleus, or wider, were equally effective; beams 10 m wide or less were less effective. The results indicate that the nuclear region is the site of the blue- and near ultraviolet-light-absorbing pigment (P_B-NUV) which mediates the timing effect of cell division. In contrast, the effect of a narrow beam of far-red (FR) light, which delays the onset of the blue-light-induced cell division, was found to be present along the entire length of the protonema cell, including the largely vacuolated basal region of the latter. Polarized FR light having the electrical vector parallel to the protonema axis was less effective than that vibrating in other directions. These observations support the hypothesis that the phytochrome controlling the timing effect is localized in the plasma membrane.     

Suppressive effect of light on the formation of DNA and on the increase of deoxythymidine monophosphate kinase activity in Chlorella protothecoides

As previously demonstrated, chlorophyll-less cells of Chlorellaprotothecoides are obtained when the alga is grown in a mediumrich in glucose and poor in a nitrogen source (urea). When thesecells are incubated in a medium enriched with a nitrogen source,there occurs, besides greening of algal cells, an active formationof DNA followed by synchronous cellular division. The DNA formationand cellular division are markedly suppressed by light of acomparatively low intensity. Blue light is most effective andred light least effective in suppression. The effect of light on the level of dTMP kinase activity inthe algal cells was investigated in relation to the photoinhibitionof DNA formation. It was found that light suppresses the increaseof dTMP kinase activity in the algal cells which starts in advanceof active DNA synthesis, and that blue light has a strongersuppressive effect than red light. ¹Present address: Institute of Medical Science, University ofTokyo, Tokyo.     

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.     

Blue Light-Induced Lethality of a Cell Wall-Deficient Mutant of the Unicellular Green Alga Chlamydomonas reinhardtii

When synchronized cultures of a cell wall-deficient Chlamydomonasreinhardtii mutant strain were grown under heterotrophic conditionsand subsequently transferred to the light, a considerable decreaseof the cell number was observed during transition to the celldivision phase. Lethality of the wall-deficient cells was inducedby blue light, but not by red or far-red light, and could notbe prevented by addition of the photosystem II inhibitor DCMU.The light-induced lethality was found to be restricted to wall-deficientcells which were agitated by bubbling with filtered air or nitrogenor vigorously shaken during the transition to the cell divisionphase. Therefore, a (blue) light-induced sensitivity to anymechanical stress seems to be the cause for cell death. In heterotrophicallygrowing cultures of the Chlamydomonas wild-type, illuminationwith blue or white light did not cause a decrease of the cellnumber but only a delay of cell divisions. The latter effectwas also observed in case of the wall-deficient mutant. Bothblue light effects are observed during the transition to thecell division phase and can be induced during the same periodof the cell cycle. Furthermore, the (blue) light-induced lethalityof wall-deficient cells was found to be prevented when the transitionto the cell division phase was inhibited by addition of antibiotics.Therefore, we assume that there is a connection between theblue light-induced sensitivity to mechanical stress and theblue light-induced delay of cell divisions. (Received September 3, 1993; Accepted November 12, 1993)     

Synchronous Cell Division in Cultured Explants of Jerusalem Artichoke Tubers: the Effects of 5-Fluorouracil on Messenger RNA Synthesis and the Induction of Cell Division

Explants of secondary xylern parenchyma tissue from Jerusalemartichoke tubers were induced to undergo cell division and de-differentiateby culture in nutrient medium. The first division was inherentlysynchronous. The system was used to study the involvement ofmessenger RNA synthesis in the induction and continuance ofcell division in previously non-dividing cells. The base analogue 5-fluorouracil (5-FU) inhibited ribosomalRNA synthesis and the processing of ribosomal RNA precursorto mature 25 S and 18 S RNAs. The synthesis of messenger-likeRNAs (heterogeneous in size, labelled to a high specific activityin a pulse incubation, and containing a polyadenylic acid sequence)was less inhibited by 5-FU. Explants grown in 5-FU did not synthesize DNA and did not divide.A direct inhibition of DNA synthesis by 5-FU added late in culturewas reversed by thymidine. An indirect inhibition of DNA synthesisoccurred when 5-FU was present from the start of culture andwas not reversed by thymidine. Because ribosomal RNA synthesisis not necessary for the induction of cell division (Fraser,1975) and because 5-FU was incorporated into mENA, probablyinterfering with its function, these results suggest that 5-FUinhibited the metabolism of mRNA which was required for DNAsynthesis and cell division. The timing of mRNA synthesis required for DNA synthesis andcell division was investigated by adding 5-FU plus thymidineto cultures at various times. By the beginning of DNA synthesisfor the first division, explants were competent, in terms ofmRNA synthesized, to complete the first division. MessengerRNA synthesis occurring before the end of the first divisionallowed explants to undergo at least three more divisions.     

RNA and protein metabolism in the particulate fractions of the gametophytes of bracken fern during growth in red and blue light

Summary The metabolism of RNA and protein in the gametophytes of bracken fern (Pteridium aquilinum) is affected by the quality of light in which they are grown. When sporelings were grown as two-dimensional gametophytes in blue light, particulate fractions separated from the sporelings exhibited greater incorporation of uridine-³H and leucine-³H into RNA and protein, respectively, than those from sporelings grown as one-dimensional protonema in red light. After various periods of exposure of gametophytes to red or blue light in the presence of uridine-³H, the nuclei-rich fraction showed the highest specific activity in RNA, and irrespective of incubation time, blue light was more effective than red light. The possibility that enhanced synthesis of RNA in the nucleus in response to blue light is significantly related to the morphological growth pattern of the gametophytes, is discussed.     

Effect of different light qualities on the ultrastructure, thylakoid membrane composition and assimilation metabolism of Chlorella fusca

Chlorella fusca (Shihira et Krauss) strain C-1.1.10 was grown under three different light qualities (red, white or blue light) in homocontinuous cultures. Under electron microscopy, blue light cultures showed enlarged cells, thinner cell walls and lower starch content than red light cells. Under blue light, the degree of stacking of the thylakoid membranes was significantly lower than under white or red light conditions. Changing the light from blue to red the ratio of exposed to appressed membranes was doubled. Compared to red light cells, blue light cells exhibited higher photosynthetic rates per chlorophyll molecule and contained less chlorophyll per dry weight. Blue light stimulated the content of soluble protein as well as that of soluble carbohydrates. The dry weight productivity per unit time was enhanced under blue light conditions. The thylakoid protein complexes which are generally assumed to be localized in the exposed membranes were found in higher concentrations under blue light than under red light. In blue light, both the Photosystem II/Photosystem I ratio and the ratio of light-harvesting chlorophyll protein to P-700 chlorophyll a -protein were lower than in red light. Blue light cells contained twice the concentration of cytochrome f , which correlates well with their higher photosynthetic capacity. When altering the light quality, the degree of change in the reaction center complexes was much lower than expected given the corresponding degree of change in the ratio of exposed to appressed membranes. These results are discussed in light of the question as to whether the variation in the stoichiometry of the laterally distributed complexes can be explained by changes in the degree of stacking alone.     

Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

The Effects of Thymidine and Its Analogues on the Differentiation of Antibody-forming Cells

Thymidine (Tdr) and its analogues 5-fluorodeoxyuridine (FUdr) and 5-bromodeoxyuridine (BUdr) inhibited the development of antibody-producing cells (PFC) in cultures of explants of rabbit spleen stimulated with sheep red cells in vitro. They were effective in this respect, even at concentrations too low to inhibit cell division.
The results indicated that Tdr had produced its effects by a selective action on DNA and RNA synthesis. In view of what is known about the mechanisms of action of Tdr and its analogues to interfere with DNA-dependent events in cells, these results supported the idea that DNA synthesis, unrelated to the needs for cell division, was required for the development of antibody-producing capacity by PFC in these cultures.     

Regulation of the Synthesis of the Lactose Repressor

Measurements of the lactose repressor over a tenfold range of cell growth rates were made on protein extracts from Escherichia coli cultures grown in media with various carbon energy sources. The concentration of lactose repressor varied with the number of genome equivalents per cell over this range in growth rates, suggesting that the number of lactose molecules within the cell is determined by the number of I gene copies present. The timing of repressor synthesis during the cell division cycle and its correlation with deoxyribonucleic acid synthesis was examined by synchronizing the cell division cycle of E. coli ED1039, in which the Lac region has been transposed from 10 to 36 min on the genetic map. Measurements of lactose repressor in the synchronized culture revealed a burst of repressor synthesis at the time of I gene duplication. The concentration of lactose repressor was found to decrease as a function of total cell protein during the division cycle until an increase in synthesis occurred, suggesting that repressor synthesis probably does not occur throughout the division cycle. A model for I gene regulation is proposed.     

Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures

Carrot cells were cultured under various light spectra and intensities at different times following the initiation of suspension cultures from callus. The highest intensity white and blue light treatments were inhibitory to growth and somatic embryogenesis. Red and green light were not different from dark treatments which produced the highest total number of embryoids. After extended time in culture, carrot cells in blue light produced secondary embryoids and anthocyanin. Cultures in red light had multiple cotyledons and orange-pigmented radicles. Leafy cotyledons occurred in all light treatments. Abscisic acid production peaked at the heart stage of embryogenesis and synthesis was most pronounced in blue light. Red light enhanced development to the heart stage. Both the red and blue light spectra may be used to manipulate carrot cell cultures to optimize growth.