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     

Variability of mitochondrial population in Chlamydomonas reinhardii

Several details have been published cocerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.     

DNA content of Phaseolus coccineus x P. vulgaris suspensors

Summary At the globular stage of embryo development, the level of DNA, as determined from microspectrophotometric analysis of Feulgen-stained squashes, was significantly higher in the interspecific hybrid suspensor than in suspensors from self-pollination of Phaseolus coccineus, the maternal parent. However, at the early-heart and early-cotyledonary stages of development, DNA content of interspecific hybrid suspensors was significantly lower than that of suspensors formed after self-pollination of either P. coccineus or P. vulgaris. The relationship between DNA content and suspensor cell length for P. coccineus and P. vulgaris at all developmental stages and between DNA content and cell area for P. coccineus at the early-cotyledonary stage was altered in hybrid suspensor cells. Nuclei in large cells of interspecific suspensors exhibited uneven distribution of polytene chromosomes and no clear nuclear outline.Paper number 18,470 of the Scientific Journal Series, University of Minnsota Agricultural Experiment Station, St. Paul, MN. This research was funded by the USDA CRGO under grant number USDA-85-CRCR-1-1676     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (S_ua). This latter cell is linked to the first by a common cell junction with the S _vn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn Sperm cell physically associated with the vegetative nucleus     

Quantitative determination of indole-3-acetic Acid in sugarbeet leaves using a double standard isotope dilution gas chromatographic assay

Quantitative levels of indole-3-acetic acid (IAA) were determined in leaf blades of two sugarbeet cultivars by a double standard isotope dilution assay using column chromatography followed by reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography with nitrogen thermionic detection. The double standard method was validated as a quantitative tool by gas chromatography/selected ion monitoring mass spectrometry using 2,′,4′,5′,6′,7′-d₅-IAA as the internal standard. Progenies of one breeding line that had been selected for a high taproot to leaf weight ratio were used to correlate IAA levels with varying leaf and plant size at day 31 from germination. In spite of size differences, no significant difference in IAA levels per unit leaf weight could be found. The possible relationship between day 31 leaves and IAA content at an earlier stage of development is discussed in the text. A second analysis used four developmental leaf stages, classified as expanding, recently mature, aging, and senescing leaves. Expanding leaves contained the most IAA, senescing leaves contained the least IAA, with recently mature leaves and aging leaves containing intermediate amounts. The DNA content of each of the four developmental leaf stages was determined and DNA levels per gram fresh weight were found to be constant at all developmental stages.     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.     

DIPLOIDY IN DNA CONTENT IN VEGETATIVE CELLS OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

DNA content of the nucleus in the placoderm desmid, Closterium ehrenbergii Meneghini was measured throughout the life cycle by epifluorescence microspectrophotometry after DNA specific dye [4′,6-diamidino-2-phenylindol (DAPI)] staining. Postulating a mean DNA content of gamete nuclei as 1C, the nucleus of a newly divided vegetative cell was 2C. Most vegetative cells in the stage of exponential growth had a DNA content from 2C to 4C, while most in stationary phase, with the highest frequency of zygote formation, were 2C. They became pre-gametes (2C) upon mixing two heterothallic strains. Four gametes were made by a DNA reduction division of each pre-gamete cell. Therefore, there was a nonmeiotic DNA reduction stage by one half. During germination, the zygote underwent meiosis to produce two gones, each of which contained one surviving nucleus (large nucleus) and one degenerating nucleus (small nucleus). The DNA content of these four nuclei was 1C basically. The DNA of the surviving nucleus duplicated to 2C and further quadruplicated to 4C without cell or nuclear division. These two 4C gones had different cell morphology from ordinary vegetative cells. After the first cell division following meiosis, each gone produced two vegetative cells in which the DNA content became 2C to 4C again.     

Changes in the polyadenylated messenger RNA population during development of Dictyostelium discoideum

The program of gene expression during the life cycle of Dictyostelium discoideum has been assessed by molecular hybridization of cDNA probes with polysomal RNA extracted at the following different stages of development: vegetative growth, interphase (2.5 hr), aggregation (8 hr), postaggregation (12 hr), and preculmination (18 hr). Several different cDNA probes were used. Two probes were prepared from vegetative stage poly(A⁺) RNA, one representing all species present and the other enriched for abundant species. A third cDNA probe was prepared from preculmination stage polysomal RNA and a fourth probe consisted of the preculmination stage cDNA depleted in those species also present at the vegetative stage. Hybridization of the various probes with the different polysomal RNA preparations has revealed developmental changes in the mRNA populations. These changes were not detected in an aggregation less mutant under similar conditions of starvation. Abundant RNA species of vegetative cells were found to drop to low levels, especially during the aggregation period. Fifty percent by mass of the RNA present in polysomes at 18 hr is not present during vegetative growth. Some of the new RNA species appeared during interphase and the remaining during the postaggregation period. A gradual increase in the number of copies per cell of certain RNA species comprising both new species as well as some shared with vegetative cells was observed throughout development. Other results indicated that the composition of polysomal and cytoplasmic RNA is similar during vegetative growth but differs markedly at 18 hr of development. Also, cytoplasmic RNA at 18 hr contained, in addition to polysomal RNA, a large proportion by mass of nonpolysomal RNA similar to vegetative RNA. The number of polysomal RNA species detected by this analysis during vegetative growth and during the preculmination stage were estimated to be 3000 and 3700, respectively. The number of copies of these RNA species ranged between 30 and 2000 per cell during vegetative growth and 3 to 300 per cell in polysomes at 18 hr. Developmentally induced RNAs which were preferentially distributed among abundant and intermediate classes were estimated to number 700–900 species.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

Life cycle of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters

Since 1995, blooms of the harmful dinoflagellate, Cochlodinium polykrikoides, have caused considerable mortality of aquatic organisms and economic loss in Korea. However, little is known about the life cycle of the species, except for the planktonic vegetative stage; therefore, the aim of this paper was to elucidate the life cycle of C. polykrikoides. Its life cycle has two morphologically different stages: an armored and an unarmored vegetative stage. Armored vegetative cells were found in seawater samples collected in late-November and developed into four-cell chained, unarmored vegetative cells under laboratory culture. In samples collected in late-May, both the armored and unarmored types (vegetative swimming stage) occurred; the former easily developed into an unarmored vegetative cell type, suggesting that the armoured–unarmored transition occurs as early as May. A presumptive resting cyst, round but folded at one side, was produced from armored type cells in laboratory conditions. It was also collected from natural bottom sediments, which suggests it is the dormant resting cyst of C. polykrikoides.     

The effect of light on DNA synthesis and related processes in synchronous cultures of Chlorella

Summary In autotrophic cultures of Chlorella synchronised by alternating light and dark periods of 16:8 hours the DNA content duplicated normally 4 times successively during the S _mphase, i. e. between the 10th and 18th hour after the beginning of the light period. This finding together with electron microscopical observations revealed that one duplication of the DNA and of the nuclei per cell proceeds every 110 minutes. All nuclei of a cell seem to undergo successive DNA syntheses and nuclear divisions synchronously. The rate of DNA synthesis was independent from illumination. On appropriate reduction of the light period the last duplication cycle fell out and the average final spore number per cell was accordingly lower.If a culture was transferred to darkness or low light intensity 3 hours before the normal end of the light period the release of spores was promoted by approximately 1 1/2 hours, provided a strong decrease of metabolically accessible carbohydrates was prevented by either an additional short illumination during the dark period or by continuing the weak light.A possible explanation for the shortening of the cell development is that, by passing over one DNA duplication and one protoplast division, the cell can enter sooner the respective subsequent developmental stages.     

Characterization of DNA synthesis during the 2-cell stage and the production of tetraploid chimeric pig embryos

The DNA content of nuclei during the 2-cell stage as well as in presumptive tetraploid embryos was investigated. In vivo produced pig zygotes were cultured to the 2-cell stage and either monitored for cleavage to the 4-cell stage or mounted at various times postcleavage and DNA content determined. The length of the 2-cell stage was 14.8 ± 3.0 hr. There was a significant increase in the length of the 2-cell stage due to the time in vitro as a zygote (P < 0.001: R² = 0.866). The DNA content increased (P < 0.05) each 2 hr postcleavage until 10 hr postcleavage. This suggested that there is a short G₁ and G₂ phase and a relatively long phase of DNA synthesis. Next, 2-cell stage embryos were pulsed with electricity to induce cell-to-cell fusion. Whereas only about half fused within 30 min (55%), most (96%) developed to the blastocyst stage. The DNA content of the nuclei of the embryos was consistent with them being tetraploid. A final experiment was designed to evaluate the ability of the tetraploid embryo to form a chimera with isolated inner cell mass (ICM) cells. Inner cell masses were isolated from d 6 embryos, cut into thirds, labeled with DiO (a membrane die) and injected into the perivitelline space of 4-cell-stage tetraploid embryos. Twelve of 17 formed blastocysts. In most 8/12), the ICM of the resulting blastocyst was labeled, whereas in one the only fluorescence was in the trophectoderm, and in two fluorescence was evenly distributed between the ICM and trophectoderm. These results suggest that it may be possible to create a fetus derived from ICM cells, or potentially stem cells, that has a tetraploid trophoblast. © 1996 Wiley-Liss, Inc.     

Karyology of a Marine Non-Motile Dinoflagellate, Pyrocystis lunula

Dinoflagellates have a unique and interesting intracellular architecture such as permanently condensed chromosomes throughout the cell cycle. However the study of dinoflagellate chromosomes is not amendable because of the unusually higher number of chromosomes and problems in sample preparation. The species of Pyrocystis spend most of their life cycle as vegetative cyst forms and have been used as experimental organisms for bioluminescence and circadian rhythms. Here, we documented the content of DNA in different life stages and the chromosome karyology in a marine non-motile dinoflagellate Pyrocystis lunula, through light and fluorescent microscopy, serial ultra-thin sectioning, and three dimension (3D) modeling. The DNA content doubles during DNA synthesis and in the end of the cell division two separate daughter cells have the approximately same fluorescent values for the mother cells. Using serial ultra-thin sectioning and 3D modeling, we report the first ultrastructural karyogram. The cells chosen were at the end of karyokinesis. A total of 98 chromosomes were counted and assigned to 49 pairs. In this species, DNA synthesis appears to occur before, or during asexual division and P. lunula lives a diplontic life cycle.     

Ultrastructural changes in maturing pollen grains ofApium nodiflorum L. (Apiaceae), with special reference to the endoplasmic reticulum

Summary The ultrastructural events in 3-cellular pollen grains ofApium nodiflorum L. are investigated during pollen maturation. Three distinct developmental stages are distinguished from the formation of sperm cells up to anthesis, whereby the rough endoplasmic reticulum (RER) is mainly involved. The most conspicious form is the highly dilated RER in the vegetative cytoplasm of the youngest pollen grains, which changes to vesicular RER in the following stage. In mature pollen grains the RER has a narrow cisternal configuration and often forms stacks. Pollen activation is preceded by the accumulation of polysaccharide particles.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

SOYBEAN GROWTH RESPONSES TO ENHANCED LEVELS OF ULTRAVIOLET-B RADIATION UNDER GREENHOUSE CONDITIONS

Soybean (Glycine max [L.] Merr. cv. Essex) was grown in an unshaded greenhouse under three levels of biologically effective ultraviolet-B (UV-B_BE) radiation (effective daily dose: 0, 11.5 and 13.6 kJ m^–2) for 91 days. Plants were harvested at regular intervals beginning 10 days after germination until reproductive maturity. Mathematical growth analysis revealed that the effects of UV-B radiation varied with plant growth stage. The transition period between vegetative and reproductive growth was the most sensitive to UV-B radiation. Intermediate levels of UV-B had deleterious effects on plant height, leaf area, and total plant dry weight at late vegetative and reproductive stages of development. Specific leaf weight increased during vegetative growth but was unaffected by UV-B during reproductive growth stages. Relative growth, net assimilation, and stem elongation rates were decreased by UV-B radiation during vegetative and early reproductive growth stages. Variation in plant responses may be due in part to changes in microclimate within the plant canopy or to differences in repair or protection mechanisms at differing developmental stages.     

Comprehensive Research on Essential Oil and Phenolic Variation in Different Foeniculum vulgare Populations During Transition from Vegetative to Reproductive Stage

Chemical composition and antioxidant activity of four fennel populations (England, Spain, Poland and Iran) were investigated during six developmental stages including two vegetative and four reproductive phases. In reproductive phase, the essential oil content of fruits decreased and the maximum content (5.9%) was obtained in immediate fruits. The essential oils were analyzed using GC/MS. trans‐Anethole was the main component of the leaves and the fruits oil. In leaves, it ranged from 41.28% in England at late vegetative stage to 56.6% in Poland population at early vegetative stage. Other major compounds of leaves were limonene, α‐pinene and (Z)‐β‐ocimene. In reproductive phases the trans‐anethole increased dramatically. It varied from 85.25% in immature fruits from Poland to 90.7% in pre‐mature stage from Spain. The highest phenolic content in the extracts at different growth stages obtained 189 mg TAE/g DW at full mature stage of seed in Iran population. The flavonoid of leaves extract ranged from 3 to 7.5 mg QUE/g DW, while in fruits extract varied from 3 to 10.3 mg QUE/g DW. Antioxidant activity of the extracts was evaluated using 1,1‐diphenyl‐2‐picrylhydrazy (DPPH) and β‐carotene model systems. Immature and full mature growth stages of fennel population from Spain indicated the highest activity in quenching of DPPH radical (74.2% and 74.5, respectively). Antioxidant activities of the extracts had high positive correlation with their phenolic contents in all fruit maturity stages. Finally, it might probably be suggested that in fennel the hot and humid condition can lead to increase trans‐anethole, while the hot and dry one can produce higher amount of phenolics and flavonoids.     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.