ANALYSIS OF STARCH CONTENT IN CHLORELLA PYRENOIDOSA (CHLOROPHYTA) BY MORPHOLOGICAL AND QUANTITATIVE TECHNIQUES

Chlorella pyrenoidosa (Chick) was grown heterotrophically in batch culture on defined medium with glucose. Morphometric analysis of cells in the exponential growth phase showed that starch accounted for 57% of the volume of the chloroplast and 36% of the total cell volume. During the stationary growth phase, the amount of starch accounted for only 36% of the chloroplast volume and 13% of the total cell volume. This represented a 36% decrease in the amount of starch/cell between the exponential and stationary phase. Determination of starch as grams/cell using quantitative techniques on cell extracts showed a comparable decrease in the amount of starch during this same transition. Based on these results, morphometric techniques provided an accurate assay of starch and have the added advantage of visualization of cellular structures not available when quantitative techniques are used.     

A specific increase in chloroplast gene mutations following growth of Chlamydomonas in 5-fluorodeoxyuridine.

Summary Growth of Chlamydomonas in the thymidine analog 5-fluorodeoxyuridine (FdUrd) leads to a reduction in the amount of chloroplast DNA and also alters the pattern of chloroplast gene transmission in crosses (Wurtz et al., 1977). We have now found that growth of Chlamydomonas in FdUrd also increases at least 10 to 20 fold the frequency of cells expressing antibiotic resistant or non-photosynthetic mutations in the chloroplast genome with no concomitant increase in nuclear gene mutations with similar phenotypes. Clearly this effect is not locus specific since the non-photosynthetic chloroplast mutations thus far isolated comprise 9 recombinationally separate loci in the chloroplast genome (Shepherd et al., 1977, 1979). Only with the use of FdUrd has isolation of this important class of non-photosynthetic mutations been possible. The efficiency of recovery of chloroplast gene mutations rises as FdUrd concentration increases from 0.1 to 1.0 mM. At higher concentrations of FdUrd, growth rates and mutant yields are reduced. We propose the analog increases the yield of chloroplast mutations by a two-step process in which mutations are first induced as a result of thymidine starvation and then become expressed because the chloroplast DNA has been greatly reduced in ploidy and possibly damaged. FdUrd has its maximal effect on both recovery of chloroplast gene mutations and chloroplast gene transmission in crosses after cells grown in the presence of the analog for several generations remain at stationary phase for about 24 h. These observations suggest that chloroplast DNA metabolism is very active in non-dividing stationary phase cells of Chlamydomonas.     

Effects of Chloroplast DNA Content on the Cell Proliferation and Aging in Chlamydomonas reinhardtii

Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4–7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Received 7 September 2000/ Accepted in revised form 14 February 2001     

Chronological transition of mitochondrial morphology in Chlamydomonas reinhardtii (Chlorophyceae) poststationary phase growth1

In Chlamydomonas reinhardtii P. A. Dangeard, mitochondrial morphology has been observed during asexual cell division cycle, gamete and zygote formation, zygote maturation, and meiotic stages. However, the chronological transition of mitochondrial morphology after the stationary phase of vegetative growth, defined as the poststationary phase, remains unknown. Here, we examined the mitochondrial morphology in cells cultured for 4 months on agar plates to study mitochondrial dynamics in the poststationary phase. Fluorescence microscopy showed that the intricate thread‐like structure of mitochondria gradually changed into a granular structure via fragmentation after the stationary phase in cultures of about 1 week of age. The number of mitochondrial nucleoids decreased from about 30 per cell at 1 week to about five per cell after 4 months of culture. The mitochondrial oxygen consumption decreased exponentially, but the mitochondria retained their membrane potential. The total quantity of mitochondrial DNA (mtDNA) of cells at 4 months decreased to 20% of that at 1 week. However, the mitochondrial genomic DNA length was unchanged, as intermediate lengths were not detected. In cells in which the total mtDNA amount was reduced artificially to 16% after treatment with 5‐fluoro‐2‐deoxyuridine (FdUrd) for 1 week, the mitochondria remained as thread‐like structures. The oxygen consumption rate of these cells corresponded to that of untreated cells at 1 week of culture. This suggests that a decrease in mtDNA does not directly induce the fragmentation of mitochondria. The results suggest that during the late poststationary phase, mitochondria converge to a minimum unit of a granular structure with a mitochondrial nucleoid.     

A Deuterium Effect in Insect Attraction

Brevibacterium flavum cells obtained from different growth phases were immobilized with κ-carrageenan and the stability of the fumarase activity was investigated. The stability of fumarase activity of the immobilized preparation of cells of the stationary growth phase was highest. The highest stability of the immobilized cells seemed to be correlated to the high stability of fumarase activity in free cells of the stationary phase. High rigidity of the cell wall and membrane of B. flavum cells of the stationary phase and firm binding of fumarase protein to the cell membrane were suggested from several lines of evidence obtained on treatment of the cells with lysozyme and detergents or sonication of the cells. Electronmicrographs showed that the cells of the stationary phase retained the original shape after repeated batch reactions. Solubilized fumarase prepared from cells of the stationary phase showed the highest stability. Experiments using the partially purified enzyme strongly suggested the existence of fumarase-stabilizing components in the cells.     

Morphological changes in the central vacuole ofBlastocystis hominis during in vitro culture

Summary Morphological changes in the central vacuole during the growth in in vitro culture ofBlastocystis hominis were investigated by light and electron microscopy. Most cells in log phase and an early stationary phase showed a positive staining reaction in the central vacuole with PAS or Sudan black B stain, whereas cells in late stationary phase showed few positive reactions. Electron microscopic observations revealed that 95% ofB. hominis cells in log phase and 50% of cells in early stationary phase, had a substantial accumulation of electron-dense material in the central vacuole. In contrast, only 25% of the organisms in late stationary phase had an electron-dense central vacuole, while more than 50% of cells had an electron-lucent central vacuole. These results indicate thatB. hominis accumulated carbohydrates and lipids in the central vacuole during cell growth and that the organism probably consumed these metabolic substances during stationary growth. Therefore, it is strongly suggested that the central vacuole is an important organelle for storage of metabolic substances, such as carbohydrates and lipids, required for cell growth.Abbreviations PBS phosphate-buffered saline - PAS periodic acid-Schiff     

CspC and CspD are essential for Caulobacter crescentus stationary phase survival

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.     

ULTRASTRUCTURE OF SPONGIOCHLORIS TYPICA DURING SENESCENCE12

The ultrastructural changes which occurred during senescence in the stationary phase of growth of the unicellular green alga Spongiochloris typica were observed. The cell wall consists of a membrane like primary wall and an inner secondary wall which becomes progressively thickened with age of the culture. During senescence the lamellae become more compact within the chloroplast. The major feature of aging is the appearance of lipid bodies which eventually come to occupy a major portion of the cell lumen. The ultrastructural changes observed to occur during senescence are discussed in relation to physiological data.     

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 .     

Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus

During exponential growth, each cell cycle of the α-purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony-forming units (cfu) steadily decrease from 10⁹ cfu ml⁻¹ to a minimum of 3 × 10⁷ cfu ml⁻¹ after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 10⁸ cfu ml⁻¹. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.     

Effects of Iron-, Manganese-, or Magnesium-Deficiency on the Growth and Morphology of Euglena gracilis1

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

Light and Electron Microscope Study of Dunaliella primolecta Butcher (Volvocida)*

SYNOPSIS. Light and electron microscope observations on Dunaliella primolecta Butcher from logarithmic and stationary phases of batch cultures are correlated. Except for the lack of a cell wall the fine structure has typical volvocid features. The transition from logarithmic to stationary phase is marked by changes in content and size of cytoplasmic vacuoles, accumulation of cytoplasmic lipid, accumulation of starch in the plastid matrix, and by the formation of autophagosome-like bodies. The organelles in stationary-phase flagellates are closely packed together because of the cytoplasmic lipid and starch-distended chloroplast. Organisms from logarithmic phase have an abundant ribosome-packed groundplasm supporting the organelles. In the cytochemical demonstration of acid phosphatase activity, Golgi cisternae and smooth and coated Golgi vesicles contain Gomori reaction product. The possible roles of the Golgi apparatus in this flagellate are discussed.     

Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum

It has been found that Dictyostelium discoideum cells from the exponential growth phase of axenically grown cultures are cohesive, whereas those from stationary phase are not. These differences in cohesiveness are seen in phosphate buffer and in axenic medium. Stationary phase medium inhibits the aggregation of log phase cells; stationary phase cells inoculated into freshly prepared medium regain their cohesiveness. Stationary phase medium may contain an inhibitor of cell cohesion. pH differences between the two types of medium are not entirely responsible for loss of cohesiveness.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Benthic flattened cells of the phylogenetically related marine dinoflagellates Protoceratium reticulatum and Ceratocorys mariaovidiorum (Gonyaulacales): a new type of cyst?

A planktonic‐benthic relationship has been described for many dinoflagellate species as part of their ecological strategy to overcome highly variable aquatic environments. Here, the phylogenetically and morphologically related marine dinoflagellates Protoceratium reticulatum and Ceratocorys mariaovidiorum were studied in relation to an unknown benthic life form. In vivo and fixed samples from cultures were analyzed in detail by light and scanning electron microscopy. In both species, a cell type with a morphology different from that of vegetative cells was observed in cultures grown until stationary phase. This cell type was always benthic, swimming sporadically only when it was disturbed. Its main feature included a strong dorsoventral compression. These cells originated from vegetative cells whose protoplasm underwent a progressive flattening, resulting in a gradual detachment of the reticulate and thick thecal plates and the formation of very thin non‐reticulated new plates with pores. When returned to fresh full‐strength medium, the cells recovered their spherical vegetative‐like morphology, including new reticulated thick plates and subsequent cell divisions. The kinetics of flattened cell formation showed that in both species, this cell type increased exponentially until the onset of the culture stationary phase and then decreased. The results of this study are discussed in the context of the planktonic–benthic coupling in dinoflagellate life cycles, including those newly appreciated to be well adapted to the benthic environment.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

Chloroplast division polarity,a marker of cell division planes during morphogenesis inBangia vermicularis Harvey (Rhodophyceae)

Summary Ultrastructural observations of dividing cells inBangia vermicularis revealed a type of chloroplast division (plastokinesis) not previously reported in the red algae. The polarity of this prekaryokinetic process serves as a reliable marker of the plane of cytokinesis, a key factor in establishing thallus morphology. At the onset of division one or more invaginations develop in the envelope of the large, lobed chloroplast and proceed centripetally through the stroma in the plane of the thylakoids, forming narrow cytoplasmic channels (CCs). The thylakoids are realigned somewhat, but are not constricted as the chloroplast is divided into two or more units. The number of resulting chloroplasts and the orientation of the CCs are dependent on cell type. Distinctive cylindrical cells at the base of the filamentous region, immediately distal to the holdfast, are shorter than broad and contain a central nucleus surrounded by a doughnutshaped chloroplast. The cylindrical morphology of the thallus is established early in the first periclinal division as multiple plastokinesis commences, generating several radially-arranged daughter chloroplasts. Cleavage of the original chloroplast is completed during subsequent cell divisions in the initial developmental stage, finally resulting in eight chloroplasts that are distributed to an equal number of wedge-shaped radial cells. Cells distal to the actively dividing basal cells are cuboidal and have a peripheral nucleus. Division of the single chloroplast prior to karyokinesis in these cells results in two or four daughter chloroplasts according to cell type. During or following plastokinesis, multilamellar bodies derived from the CE appear to serve as the source of membranes for the developing septum in the channels. Septa link to proliferations of the plasmalemma in areas of slight cell wall (CW) indentations, and are completed between daughter nuclei after karyokinesis, producing a cleavage channel. Subsequently, primary CW material is deposited between the two septal membranes. The shape and arrangement of daughter cells in each of the developmental stages in the thallus are defined by the planes of cell division. These are indicated by both the orientation of CCs and the polar orientation of nuclear division which is always at right angles to the CC.Abbreviations CC cytoplasmic channel - CE chloroplast envelope - CW cell wall - ER endoplasmic reticulum - MLB multilamellar body