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 .     

Induction and regulation of chloroplast replication in mature tobacco leaf tissue

Chloroplast replication was induced in mature tobacco leaf tissue (Nicotiana tabacum L.) by culturing leaf discs on a sterile medium composed of salts and sucrose. Chloroplast replicaton is greatly enhanced by the addition of kinetin to this medium. Kinetin also enhances cell enlargement, but cell division does not occur. Chloroplast replication is nonsynchronous and proceeds most rapidly when the cell enlargement rate decreases. Chloroplast replication is light-dependent, but cell enlargement occurs in both light and dark. Chloroplast replication resumes when discs cultured in the dark are returned to the light. It appears that chloroplast replication is related to cell expansion. The possibility of inducing synchronous replication of chloroplasts in tobacco cells is discussed.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

Drifts in DNA level in the cyanobacterium Anacystis nidulans during synchrony induction and in synchronous culture

Cultures of the cyanobacterium Anacystis nidulans were synchronized by using alternating light-dark cycles. The DNA level in the cells was determined, at intervals, during pre-synchrony treatment and subsequent synchronous growth. The DNA content/cell gradually increased during synchrony induction and reached a maximum value after about 9–10 dark-light cycles, coinciding with the minimum length of pre-synchrony treatment necessary for obtaining good synchrony of cell division in our system. DNA synthesis was found to be discontinuous in the synchronous cultures. The results suggest two gaps in DNA synthesis, one occurring before and one after cell division. The results are compared with the relevant data published on the life cycle of other prokaryotic microorganisms.     

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     

Chloroplast proliferation based on their distribution and arrangement inAdiantum protonemata growing under red light

Chloroplast proliferation was investigated inAdiantum protonemata growing under continuous red light. Cell division is absent when cells are grown under red light. The chloroplast number increases as the cell length increases, therefore the chloroplasts divide in the absence of cell division. Chloroplasts in the basal part of the filamentous protonemal cell migrate gradually toward the cell apex, but there is no large net migration from the tip to the base or vice versa, indicating that chloroplast division takes place in the apical part of the protonemata. Chloroplast number in the apical 100 μm was maintained at about 200 during cell growth at least over eight days. The chloroplasts were either dumbbell- or ellipsoid-shaped. Dumbbell-shaped chloroplasts are abundant everywhere in a protonema, ranging from 30 to 50% of the total chloroplasts. The dumbbell-shaped chloroplasts attached to or very close to the plasma membrane seem to be the ones that are dividing but the dumbbell-shaped ones in the other regions do not divide. These data support the hypothesis that a signal from the plasma membrane induces the dumbbell-shaped chloroplasts to divide.     

arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts

A novel mutant of Arabidopsis thaliana, arc6 (accumulation and replication of chloroplasts), has been isolated from a transfer DNA-mutagenized population of Arabidopsis seedlings. arc6 has the most extreme arc mutant phenotype we have yet described, with only one to three chloroplasts per leaf mesophyll cell compared to a mean of 83 in cells of the wild-type var Wassilewskija. The chloroplasts of arc6 are 20-fold larger than wild-type chloroplasts.Chloroplast division is almost certainly precluded in arc6 mesophyll cells, since chloroplast number per cell does not increase during mesophyll cell expansion. arc6 chloroplasts are long and thin in cross-section and only one-half the width of wild-type chloroplasts and the arrangement of thylakoid membranes is largely unaltered. arc6 segregates as a monogenic recessive nuclear mutation in a normal Mendelian manner and the arc6 phenotype is stably inherited for at least four generations. arc6 plants grow normally and are fertile, although the rosette leaves appear curled and twisted. arc6 plants accumulate 70 to 75% of the biomass of wild type. The phenotype of this novel mutant is discussed in relation to the nature of the control of chloroplast division in leaf cells.     

Vitamin B12 and the macromolecular composition of Euglena. I. Kinetic analysis of the cell cycle and chloroplast replication

The kinetics of cell division, chloroplast replication and mean DNA/cell of cultures progressing through B₁₂ deficiency do not follow smooth curves, but contain transient plateaus which are consistent mathematically with the hypothesis that one portion of the Euglena cell cycle, the S phase, is differentially extended under B₁₂ deficiency. A computer simulation of the B₁₂-deficient cultures was capable of duplicating the division kinetics of the actual culture. Chloroplast replication in B₁₂-deficient cells is not directly affected by B₁₂ deficiency, but is a function of the division kinetics of the cells. The chloroplasts continue to replicate initially at a high rate after the cells have entered B₁₂ deficiency, and then follow the kinetics of the cells.     

Synchrony of Nuclear Replication in Individual Hyphae of Aspergillus nidulans

The synchrony of nuclear replication in individual, multinucleate hyphae of Aspergillus nidulans has been investigated. Samples were taken from cultures of germinating conidiospores, and the relative frequency of hyphae containing two to eight nuclei was determined. Because the conidiospores are mononucleate, complete synchrony will yield populations of hyphae containing only 2ⁿ nuclei, n being the number of doublings after germination. The appearance of hyphae with total numbers of nuclei other than 2ⁿ will indicate lack of synchrony. The relative frequency of hyphae not having 2ⁿ nuclei will depend on the degree of synchrony in the individual hyphae; numerical aspects of this relation are discussed. In two different strains, replication of the nuclei in any one hypha was highly synchronized when the dry weight doubling time was 1.4 to 1.8 hr. As the doubling time was made longer by changing the nitrogen or carbon source, synchrony was progressively lost. At the slowest growth rate tested, the interval between the division of the fastest and the slowest nucleus equaled 48% of the dry weight doubling time. The active replication of some nuclei in a hypha where other nuclei were resting suggested that nuclear duplication in this eukaryotic organism may be controlled by specific initiators.     

The influence of the slowing of Earth's rotation: A hypothesis to explain cell division synchrony under different day duration in earlier and later evolved unicellular algae

Every year the Earth's rotation period is reduced, mainly due to the tidal drag of the moon. The length of day increases continuously by about 1 h every 200 million years. The period of rotation around the Sun remains constant; hence, the length of the year remains constant, so years acquire progressively fewer days. Many unicellular algae show rhythmicity in their cell division cycle. If primitive algae evolved under a shorter day duration, then it is possible that the early-evolved algae had to synchronize their cell division cycle to shorter lengths of day than did later-evolved algae. We tested this hypothesis by growing Cyanobacteria, Dinophyceae, Prasinophyceae, Bacillariophyceae and Conjugatophyceae (evolutionary appearance probably in this order) at 8∶8 h light-dark cycles (LD), 10∶10 LD, and 12∶12 LD, at 20 or 27°C. Cyanobacteria synchronized their cell division cycles optimally at 8∶8 h LD, Dinophyceae and Prasinophyceae at 10∶10 h LD, and Conjugatophyceae and Bacillariophyceae at 12∶12 h LD. The synchrony of cell division was scarcely affected by temperature. Results suggested that the early evolved unicellular autotrophic organisms such as the Cyanobacteria synchronized their cell division cycle under a shorter day duration than later-evolved unicellular algae, and these traits may have been conserved by quiescent genes up to the present day.     

CULTURE STUDIES ON THE MARINE GREEN ALGA HALICYSTIS PARVULA—DERBESIA TENUISSIMA. II. SYNCHRONY AND PERIODICITY IN GAMETE FORMATION AND RELEASE

Unialgal cultures of the macroscopic, vesicular, coenocytic gametophyte (Halicystis parvula Schmitz) of Derbesia tenuissima (DeNotaris) Crouan fr. were grown under various environmental regimes to elucidate the cytology of gamete formation and the factors controlling synchronous gamete formation and release. No synchrony of nuclear division was observed in vegetative plants or during the early stages of gamete formation. In the later stages of gamete formation in plants in a light-dark cycle, nuclear divisions within any gametangium were synchronous, and the stages of gamete formation were synchronous for the population. This synchrony was not as great for plants in continuous light. Gametes of plants in a light-dark cycle were released explosively immediately following the dark-to-light transition. Release was random and much less forceful for plants in continuous light. After a certain stage of gamete formation, gamete release was timed to occur after a particular interval of darkness, but release could be triggered by light during the last portion of this interval. The length of the dark interval was shorter for male plants than for females, but the period of light sensitivity was longer for females. Formation of gametangia by series of isolated plants was also synchronous and sometimes periodic under certain conditions. Intervals between gametangia on the same plant varied from 2 to 14 days but were usually 4 or 5 days (unlike plants in nature, which show a bi- or tri-weekly periodicity). Male and female plants did not differ in synchrony or periodicity. Different media affected the number of gametangia formed over a period of time but not the synchrony of formation. Under some conditions changing the medium had a stimulating or synchronizing effect. Non-repeated temperature changes also synchronized gamete formation. Optimum temperature for continued gamete formation was about 21 C. Regular daily light and temperature variation together maintained synchronous and periodic gamete formation in populations of isolated plants. Reproduction diminished and became less synchronous at constant temperature either in continuous light or under a light-dark schedule, although in the light-dark regime steps in the formation of any given gametangium remained synchronous with the light-dark cycle. Length of times between gametangial formation on individual plants showed a tendency to occur in multiples of the usual period lengths; e.g., plants sometimes tend to “skip” intervals, thus maintaining the synchrony of the population. These results suggest that interaction between daily environmental cycles and an endogenous physiological cycle may maintain periodic reproduction.     

SYNCHRONOUS GROWTH IN THE COLONIAL EUDORINA ELEGANS (CHLOROPHYCEAE)1,2

Eudorina elegans Ehrenberg has been synchronized by growing the organisms at 32 C in an enriched medium on a 16:8 LD cycle. Nuclear division occurred during the light period and 2–4 h was required, for the population to complete the sequence of 4 or 5 divisions each cell normally undergoes. Each cell division required 2–3 min with ca. 15 min as the intermitotic time. The sequence was repealed, every 24 h as long as synchrony was maintained.     

Analysis of the schedule of DNA replication in heat-synchronized Tetrahymena

The temporal schedule of DNA replication in heat-synchronized Tetrahymena was studied by autoradiographic and cytofluorometric methods. It was shown that some cells, which were synchronized by selection of individual dividing cells or by temporary thymidine starvation, incorporated [³H]thymidine into macronuclei in a periodic fashion during the heat-shock treatment. It was concluded that supernumerary S periods occurred while cell division was blocked by high temperature. The proportion of cells which initiated supernumerary S periods was found to be dependent on the duration of the heat-shock treatment and on the cell cycle stage when the first heat shock was applied. Cytofluorometric measurements of Feulgen-stained macronuclei during the heat-shock treatment indicated that the DNA complement of these cells was substantially increased and probably duplicated during the course of each S period. Estimates of DNA content also suggested that the rate of DNA synthesis progressively declined during long heat-shock treatments. These results indicate that the mechanism which brings about heat-induced division synchrony is not an interruption of the process of DNA replication. Further experiments were concerned with the regulation of DNA synthesis during the first synchronized division cycle. It was shown that participation in DNA synthesis at this time increased as more cells were able to conclude the terminal S period during the preceding heat-shock treatment. It is suggested that a discrete period of time is necessary after the completion of DNA synthesis before another round of DNA synthesis can be initiated.     

Interpretations on chloroplast reproduction derived from correlations between cells and chloroplasts

Summary The size ranges of chloroplasts in living mesophyll cells of Spinacia oleracea, Allium cepa, Beta vulgaris (Swiss chard and red beet) and Nicotiana glutinosa are extremely wide, e.g., ranging from about 6 µ² to 103 µ² in face area for spinach. Moreover, the size distributions are positively skewed. We interpret the size range and skewed size distributions primarily to reflect an enormous growth of the bulk of the chloroplasts from small, equal-sized chloroplasts produced by fission of a small sub-population of constricted mature chloroplasts. While actual fission has never been observed, a slow division rate of the constricted chloroplasts in N. glutinosa can account for the increase in chloroplast numbers per cell during leaf development and for the presence of small, non-constricted chloroplasts after the small chloroplasts which developed during the initial meristem activity have enlarged. Chloroplast numbers and total amount of chloroplast material per cell face were positively correlated with mesophyll-cell face size. However, the fraction of the cell face occupied with chloroplasts was essentially constant and independent of cell size and cell age while being markedly different for different species of plants. There appear to be some family characteristics in that closely related species have similar size-distributions and ranges of chloroplast sizes. The observations are discussed with respect to the ontogeny of chloroplasts in higher plants.     

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole     

A Diurnal Settling Rhythm in Platymonas subcordiformis Hazen*

SYNOPSIS. In cultures of Platymonas subcordiformis Hazen, grown in appropriate light-dark cycles, as many as 75% of the cells adhered to the surface of the glass culture vessel toward the end of the light period of each day. Cell division occurred primarily while the cells were attached. Subsequently, motile daughter cells were released into the growth medium by the rupture of the mother cell theca. The settling behavior appears to be an integral part of the life cycle being synchronized to the same extent as cell division.     

Gibberellin indirectly promotes chloroplast biogenesis as a means to maintain the chloroplast population of expanded cells

Chloroplast biogenesis needs to be well coordinated with cell division and cell expansion during plant growth and development to achieve optimal photosynthesis rates. Previous studies showed that gibberellins (GAs) regulate many important plant developmental processes, including cell division and cell expansion. However, the relationship between chloroplast biogenesis with cell division and cell expansion, and how GA coordinately regulates these processes, remains poorly understood. In this study, we showed that chloroplast division was significantly reduced in the GA‐deficient mutants of Arabidopsis (ga1‐3) and Oryza sativa (d18‐AD), accompanied by the reduced expression of several chloroplast division‐related genes. However, the chloroplasts of both mutants exhibited increased grana stacking compared with their respective wild‐type plants, suggesting that there might be a compensation mechanism linking chloroplast division and grana stacking. A time‐course analysis showed that cell expansion‐related genes tended to be upregulated earlier and more significantly than the genes related to chloroplast division and cell division in GA‐treated ga1‐3 leaves, suggesting the possibility that GA may promote chloroplast division indirectly through impacting leaf mesophyll cell expansion. Furthermore, our cellular and molecular analysis of the GA‐response signaling mutants suggest that RGA and GAI are the major repressors regulating GA‐induced chloroplast division, but other DELLA proteins (RGL1, RGL2 and RGL3) also play a role in repressing chloroplast division in Arabidopsis. Taken together, our data show that GA plays a critical role in controlling and coordinating cell division, cell expansion and chloroplast biogenesis through influencing the DELLA protein family in both dicot and monocot plant species.