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     

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION1

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’     

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.     

Protective effect of sodium selenite on ofloxacin-induced loss of chloroplast DNA inEuglena gracilis

Some xenobiotics induce permanent loss of chloroplasts inEuglena gracilis which results in the growth of white mutant colonies (bleaching effect). The effect of ofloxacin and sodium selenite on the organization of chloroplast DNA ofE. gracilis was studied by fluorescent microscopy. In the presence of ofloxacin DNA-specific staining with DAPI revealed a decrease in chloroplast DNA content and in the number of nucleoids per chloroplast. This was accompanied with a decrease of chloroplast number per cell and with the loss of stigma. Spectrophotometry of chlorophyll extract revealed changes in th ratio of chla: chlb. The presence of sodium selenite protected chloroplast DNA against the inhibitory effect of ofloxacin.     

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.     

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6-diamidino-2-phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp-nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread-like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread-like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI-stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.     

Loss of chloroplastic DNA in a Euglena mutant during growth in darkness

The mutant strain U of Euglena gracilis, different from the wild type strain Z, has lost the ability to form chloroplasts during growth in the dark.Chloroplastic DNA could not be detected by CsCl density analysis in the dark-grown strain U. Chloroplast nucleoids fluoro-stained by DAPI were found in the light-grown cells, but not in the dark-grown U. Target number analysed by UV irradiation on the chloroplast formation ability decreased rapidly during cell-growth in darkness. These results suggest that U has lost plastid DNA during cell-growth in darkness.Abbreviations DAPI 4,6-diamidino-2-phenylindole - PSI and PSII photosystems I and II - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Visualization by fluorescence of chloroplast DNA in higher plants by means of the DNA-specific probe 4'6-diamidino-2-phenylindole

The DNA in isolated chloroplasts was visualized by the fluorescent probe 4'6-diamidino-2-phenylindole (DAPI). When excited with light of 360 nm, the DNA-DAPI complex fluoresces brilliantly at 450 nm. Nuclei also fluoresce but their nucleoli do not. RNase and Pronase treatment of chloroplasts did not affect the fluorescence but both pre- and posttreatment of DAPI-stained chloroplasts with DNase specifically destroyed the fluorescence. DNA-DAPI complexes in the chloroplasts show up as bright dots. These are distributed uniformly within the chloroplast except for the outer margins. The fluorescent dots can be seen at different focal levels. The number of DNA dots is roughly proportional to chloroplast area which, in turn, is a function of leaf size. The number of fluorescent dots also gave the impression that large leaves with large chloroplasts contain more chloroplast DNA than nuclear DNA.     

金黄滴虫叶绿体拟核的荧光镜检及电镜观察

金黄滴虫细胞在用DNA特异的荧光染料DAPI处理后,在荧光显微镜下细胞核和叶绿体拟核均散发蓝色荧光,穗晰可见。每一叶绿体有一拟核,拟核沿叶绿体的周缘排列,形状相当于叶绿体的轮廓,成不规则的两叶形环。环的全长约在20—30υm之间。 拟核环大多是单线的,有些拟核环出现或短或长的双线部分,有时甚至几乎整个拟核环都可变为双线。这表明拟核环通过“纵裂”而形成双环,在叶绿体分裂时,分别进入两个子叶绿体。这一情况在电镜照片上得到了证实。 叶绿体分裂和细胞分裂之间似乎不存在严格的相关性,这是导致细胞中叶绿体数目多于1个的原因。     

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     

Acetabulaira mediterranea: circadian rhythms of photosynthesis and associated changes in molecular structure of the thylakoid membranes.

Acetabularia mediterranea algae, grown in three different light-dark regimes, were frozen in liquid nitrogen at c.t.(1) 0 and c.t. 6 and a record made of 77 degrees K fluorescence emission spectra of their chloroplasts. Algae grown under LD cycles exhibited a clear circadian rhythm of oxygen production. The low temperature fluorescence emission spectrum at c.t.0 was different from that at c.t.6 and this difference was increased by submitting the algae to successive "freeze-thaw" treatment. Similar results were obtained in DD, and the photosynthesis rhythm remained fully expressed. Algae grown in LL, where no rhythm of photosynthesis could be detected in the samples because there is a great individual variability in period lenght under these conditions, exhibited a similar difference in their low temperature flourescence emission spectra between c.t.0 and c.t.6. We conclude that the circadian rhythm in low-temperature fluorescence emission of the chloroplasts in Acetabularia is related to the circadian rhythm in photosynthesis.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

A Suppressor of a Mating-Type Limited Zygotic Lethal Allele Also Suppresses Uniparental Chloroplast Gene Transmission in Chlamydomonas Monoica

Uniparental inheritance of Chlamydomonas chloroplast genes is thought to involve modification of maternal (mt(+)) chloroplast genomes to protect against a nuclease that is activated after gamete fusion. The mating-type limited mtl-1 mutant strain of Chlamydomonas monoica is unable to protect mt(+)-derived chloroplast DNA. Zygotes homozygous for mtl-1 lose all chloroplast DNA and fail to germinate. We have selected for suppression of this zygote-specific lethality, and have obtained 20 mutant strains that produce viable homozygotes despite the continued presence of the mtl-1 allele. Genetic analysis indicates that the suppressor mutations are all recessive alleles at a single locus (sup-1) which is unlinked to mtl-1. Crosses between sup-1 strains carrying distinctive chloroplast antibiotic resistance markers also show predominantly biparental chloroplast gene transmission. Chloroplast nucleoids of both parental origins (stained with the DNA-specific fluorochrome, DAPI) are retained in the zygotes homozygous for sup-1. The data are compatible with the idea that the sup-1 (suppressor of uniparental inheritance) locus may encode a chloroplast DNA nuclease that is expressed from both parental genomes.     

Localization of plasmidlike DNA in giant-celled marine green algae

Summary Novel extrachromosomal DNA molecules were localized in giant-celled marine green algae by organelle isolation and fluorescence in situ hybridization methodologies. Nucleic acids extracted from isolated chloroplasts ofErnodesmis verticillata andVentricaria ventricosa were greatly enriched in plasmidlike DNA species. Fluorescence in situ hybridization was employed to resolve further the subcellular location of these molecules. Cloned restriction fragments of the algal plasmidlike DNA hybridized solely to low-molecular-weight DNA in Southern blots; they did not hybridize to any chromosomal DNA. Probes were generated from these clones that either did (Northern-positive) or did not (Northern-negative) hybridize to RNA species in Northern blots. Probes specific for localizing the plasmidlike DNA were generated from the latter clones, whereas probes potentially localizing both DNA and relevant mRNA species were generated from the former ones. After hybridization and signal amplification via indirect immunofluorescence, fluorescent punctae were visible surrounding the single pyrenoid in each chloroplast with both types of probes. The punctae were arranged in a hollow spherical configuration, as resolved by confocal laser scanning microscopy. Nearly twice as many punctae per chloroplast were present inV. ventricosa (11.5) as there were inE. verticillata (6.0). The differential distribution of plasmidlike DNA within each chloroplast was in contrast to chloroplast chromosomal DNA, which occurred as multiple nucleoids scattered throughout the entire organelle. The localization of plasmidlike DNA within chloroplasts correlates well with previous sequence data indicating that these molecules contain putative open reading frames encoding protein components of photosystems I and II.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylmdole - FITC fluorescein isothiocyanate - FISH fluorescence in situ hybridization - HMW high molecular weight - LMW low molecular weight - ORF open reading frame     

Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.

Mitochondrial nucleoids (mt-nucleoids) of the A2780 line of cultured human cells were stained with DAPI and observed using an epifluorescence microscope. The mt-nucleoids appeared to be organized compactly in mitochondria. Numbers of mt-nucleoids per mitochondrion ranged from 1 to more than 10, and 70% were "multinucleated" mitochondria. Intensities of fluorescence of mt-nucleoids in each mitochondrion were measured by a video-intensified microscope system (VIM system) and copy numbers of mitochondrial DNA (mtDNA) in each mitochondria were determined. The copy numbers of mtDNA per mitochondrion ranged from 1 to 15, and the average was 4.6. Because the cells had 107 mitochondria on average, the copy number of mtDNA per cell was estimated to be about 500.