Sigrid Berger

Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis

Exopolysaccharide (EPS) synthesis by Erwinia amylovora depends on environmental and genetic predispositions. To measure the amount of the acidic EPS amylovoran synthesized by E. amylovora cell cultures, a turbidity assay using cetylpyridinium salt was developed. The EPS produced by bacteria grown on solid media was additionally characterized by its water content. The amylovoran capsules were visualized in situ by staining with fluorescein isothiocyanate (FITC)-labelled lectin from Abrus precatorius, which reacts with the galactose residue of the EPS side chain. The staining and the turbidity assays were applied to suspension cell cultures or to cells from colonies and did not require any purification steps. Lectin staining was superior to electron microscopic (EM) techniques for visualization of capsules. For EM, the capsule was stabilized with polycationic ferritin. In contrast to lectin staining, only a small fraction of the cells was found to be EPS-coated in the EM assay. An increase in capsulation and in amylovoran production was found in conjunction with mutations in a ribosomal protein conferring resistance to streptomycin. Furthermore, the presence of sorbitol in the growth environment resulted in high synthesis of amylovoran. Cells in the stationary growth phase continued to produce amylovoran. Apparently, the strong dependence of the fireblight pathogen on capsules requires the capacity for EPS synthesis in all growth stages in order to escape plant defence reactions.

Morphology of the nucleo-cytoplasmic interactions during the development ofAcetabularia cells

SummaryThe ultrastructure of the growing and maturing primary nucleus ofAcetabularia mediterranea andAcetabularia major has been studied with the use of various fixation procedures. Particular interest has been focused on the details of the nuclear periphery and the perinuclear region. It is demonstrated that early in nuclear growth a characteristic perinuclear structural complex is formed which is, among the eukaryotic cells, unique toAcetabularia and related genera. This perinuclear system consists essentially ofa)the nuclear envelope with a very high pore frequency and various pore complex associations with granular and/or threadlike structures some of which are continuous with the nucleolus;b)an approximately 100 nm thick intermediate zone densely filled with a filamentous material and occasional small membraneous structures from which the typical cytoplasmic and nuclear organelles and particles are excluded;c)an adjacent lacunar labyrinthum which is interrupted by many plasmatic junction channels between the intermediate zone and the free cytoplasm;d)numerous dense perinuclear bodies in the juxtanuclear cytoplasm which are especially frequent at the junction channels and reveal a composition of aggregated fibrillar and granular structures;e)very dense exclusively fibrillar aggregates which occur either in association with the perinuclear region of the lacunar labyrinthum or, somewhat further out, in the cytoplasmic strands between the branches of the lacunar labyrinthum in the form of slender, characteristic rods or “sausages”. A variety of other membraneous and non-membraneous structures characteristic of the juxtanuclear cytoplasm is described. The organization of the individual components in this special complex is summarized in a model drawing. The dynamic and transitory nature of this perinuclear complex apparatus is emphasized and its possible role in nuclear functions and in regulating nucleocytoplasmic interactions is discussed.

18S RDNA AND EVOLUTION IN THE DASYCLADALES (CHLOROPHYTA) - MODERN LIVING FOSSILS

Phylogenetic relationships were inferred from parsimony and distance analyses of nuclear small‐subunit ribosomal DNA sequences taken from 14 species representing 8 of the 11 extant genera in the Dasycladales. Of 1733 aligned positions, 412 (23.8%) were variable and 251 (61%) of those were phylogenetically informative within the Dasycladales. Secondary structure was analyzed and taken into account during all phases of data analysis. Robustness of the trees was assessed using bootstrap analysis and g1 statistics of tree‐length decay. Strongly supported branches were robust to all methods of analysis regardless of weighting schemes used. The secondary structure of the 18S within the Dasycladales agrees with that of other green algae with the exception of a shared deletion in stemloop E10‐1 (ca. 13 nucleotides long), which provides additional support for the uniqueness of this monophyletic group. A molecular clock was calibrated from the dasyclad fossil record and suggests a radiation of the Acetabulariaceae at 120 ± 30 million years (Ma) ago and the Dasycladaceae 215 ± 40 Ma ago. The split of the two lineages from a shared ancestor is estimated at 265 ± 50 Ma ago. Within the Dasycladaceae, Neomeris and Cymopolia are sister taxa, as are Batophora and Chlorocladus. Bornetella groups with the Neomeris and Cymopolia clade in 78% of the bootstrap replicates. Relationships among the Acetabulariaceae show that Acetabularia and Polyphysa do not form monophyletic groups as presently circumscribed. No evidence indicates that Acicularia is the oldest genus. Halicoryne, Chalmasia, and Dasycladus were not included in the analysis. Molecular data provide afresh background perspective from which to discuss the evolution of one of the most ancient lineages of green plants.

Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome

SummaryMesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca++/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.

RNA-synthesis in Acetabularia. I. RNA-synthesis in enucleated cells.

SummaryIncorporation of precursors of RNA into the enucleated cells ofAcetabularia mediterranea andPolyphysa cliftonii has been studied under conditions which exclude the possibility of errors due to contamination of the preparations by nuclei, nuclear debris or microorganisms.Radioactive RNA has been isolated from the chloroplast, mitochondrial and supernatant cytoplasm fractions of nucleated and enucleated cells. Sucrose density gradient centrifugation of the isolated labelled RNA produces a sedimentation profile of radioactivity which is similar to that of RNA isolated fromE. coli ribosomes.Anion exchange chromatography of alkaline hydrolysates of the 23 s and 16 s ribosomal RNA fractions shows incorporation of labelled uracil into RNA in the form of 2′(3′)-UMP and 2′(3′)-CMP. Labelled guanosine is incorporated only as 2′(3′)-GMP.A slowly sedimenting radioactivity peak has been chromatographed on a Hershey column and found to correspond to cold t-RNA.

An apparent lack of non-transcribed spacers in rDNA of a green alga

SummaryActively transcribed DNA was made visible in nuclear material from the giant unicellular green alga Batophora oerstedii (Chlorophyta, Dasycladaceae) by means of Millers spreading technique. A conspicuous feature of a major part of the spread material is that actively transcribed regions are adjacent to each other resulting either in a lack of, or at least in extremely short spacer DNA.The length and the localization of these repetitive transcriptional units in the nucleolar material indicate that they are rRNA cistrons. In addition to the spacer-free DNA, repetitious structures have been found within the nucleolar material of individual nucleic, in which spacers occur. The transcriptional units in the spacer-free regions appear to have the same length as the transcriptional units separated by spacers.

18S rDNA phylogeny and evolution of cap development in Polyphysaceae (formerly Acetabulariaceae; Dasycladales, Chlorophyta)

Abstract Cells of the members of the Dasycladales have a unique body plan well known from fossils. They persist today in 38 recognized species. This study investigates in detail the development of reproductive structures in 17 Polyphysaceae (= Acetabulariaceae) species and provides a molecular phylogenetic analysis of 18S ribosomal DNA sequence data of 23 species of the order Dasycladales, including 17 of the 19 extant members of the family Polyphysaceae. Reproductive cap development is documented by scanning electron microscopy in 17 species, by histological sections in five species, and by growth measurements. Other morphometric data are also provided for most species. Bayesian analysis of DNA data reveals three well-supported clades for which morphological synapomorphies exist, but which are not completely in accordance with previous generic concepts. An early-branching clade comprising the monotypic Chalmasia and two species of Halicoryne is characterized by the formation of buds by the pointed cap rays. The second clade, comprising five species of Polyphysa (except P. peniculus), is characterized by corrugation of the cell stalk. It forms a sister group to the third clade, which comprises all eight Acetabularia species, plus P. peniculus and Acicularia schenckii. This clade is united by the lack of a velum covering the developing caps and by the relative timing of cap ray initiation. The extended Acetabularia clade is also characterized by the presence of a corona inferior, a structure that apparently was secondarily reduced in P. peniculus. Acetabularia acetabulum, a model species for cell biology, is sister to all other species of this clade. Its unique, congenitally fused cap rays may have evolved by a simple shift of growth zones during cap initiation. We propose to include both the monotypic Acicularia (as Acetabularia schenckii) and Polyphysa peniculus (as Acetabularia peniculus) in the genus Acetabularia. For the remaining Polyphysa species of clade 2, a new genus name, Parvocaulis S. Berger et al., is proposed.

ULTRASTRUCTURE DURING DEVELOPMENT OF THE NUCLEUS OF BATOPHORA OERSTEDII (CHLOROPHYTA; DASYCLADACEAE)1

The primary nucleus of Batophora oerstedii J. Agardh like that of the related Acetabularia, undergoes a great increase in size throughout vegetative development. Formation of small secondary nuclei represents an irreversible stage in the development of reproductive structures in the gametangia. Changes observed in the course of the life cycle include: i) an increase from 3 to 200 μm diam, of the nucleus; ii) increase in number of nucleoli; iii) development of the perinuclear region; iv) increased pore density in the nuclear membrane; v) development of chromosomes in the nucleoplasm; vi) formation of secondary nuclei; and, vii) division of secondary nuclei.

80 S ribosomes inAcetabularia major redundancy of rRNA cistrons

SummaryHigh redundancy of rRNA cistrons inAcetabularia has been postulated from an estimation based on the rate of synthesis and on the long half life of rRNA in this organism. This hypothesis was proved inAcetabularia major by means of Millers spreading technique. The rRNA cistrons in this giant single cell alga have been shown to be highly redundant. The redundancy seems to be correlated to the length of the cell indicating that the amplification takes place during at least the greater part of the vegetative phase. Up to 52 cistrons have been detected on one DNA matrix. The length of the rRNA cistron corresponds quite closely to what would be expected for a DNA coding for RNA of the molecular weight 2.1×106 daltons. This value indicates that there is little if any loss of nucleotide material during the maturation of the 18 S and 26.5 S rRNAs inAcetabularia.

Ribosomal DNA in different members of a family of green algae (Chlorophyta, Dasycladaceae): an electron microscopisal study.

SummaryActive cistrons and non-transcribed spacers of rDNA were made visible in spread preparations of nucleolar material from different species of the green alga family Dasycladaceae (Chlorophyta). The data observed show a difference in length in transcribed rDNA regions between Acetabularia peniculus Solms-Laubach, Acetabularia dentata Solms-Laubach on the one hand and Acetabularia ryukyuensis Okamura et Yamada, Dasycladus clavaeformis J. Agardh, Cymopolia van bosseae Solms-Laubach and Batophora oerstedii J. Agardh on the other: The large size difference between spacer intercepts of different species is very conspicuous. While the Acetabularia species have long non-transcribed spacers they are very short or even not observable in the other Dasycladaceen species studied. Peculiarities found in rDNA include extra long spacers between cistrons, single extra long cistrons together with rDNA cistrons on the same strand and rDNA-sized cistrons alternately arranged on one strand with larger sized cistrons. In spite of extensive searching, inversions were never found in spread nucleolar material. However, they were frequent in spread material of whole nuclei.