Ingrid Capesius

Plant Mitochondrial RNA Editing

Abstract. RNA editing affects messenger RNAs and transfer RNAs in plant mitochondria by site-specific exchange of cytidine and uridine bases in both seed and nonseed plants. Distribution of the phenomenon among bryophytes has been unclear since RNA editing has been detected in some but not all liverworts and mosses. A more detailed understanding of RNA editing in plants required extended data sets for taxa and sequences investigated. Toward this aim an internal region of the mitochondrial nad5 gene (1104 nt) was analyzed in a large collection of bryophytes and green algae (Charales). The genomic nad5 sequences predict editing in 30 mosses, 2 hornworts, and 7 simple thalloid and leafy liverworts (Jungermanniidae). No editing is, however, required in seven species of the complex thalloid liverworts (Marchantiidae) and the algae. RNA editing among the Jungermanniidae, on the other hand, reaches frequencies of up to 6% of codons being modified. Predictability of RNA editing from the genomic sequences was confirmed by cDNA analysis in the mosses Schistostega pennata and Rhodobryum roseum, the hornworts Anthoceros husnotii and A. punctatus, and the liverworts Metzgeria conjugata and Moerckia flotoviana. All C-to-U nucleotide exchanges predicted to reestablish conserved codons were confirmed. Editing in the hornworts includes the removal of genomic stop codons by frequent reverse U-to-C edits. Expectedly, no RNA editing events were identified by cDNA analysis in the marchantiid liverworts Ricciocarpos natans, Corsinia coriandra, and Lunularia cruciata. The findings are discussed in relation to models on the phylogeny of land plants.

The origin of land plants : phylogenetic relationships among charophytes, bryophytes, and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

Complete nuclear-encoded small-subunit 18S rRNA (=SSU rRNA) gene sequences were determined for the prasinophyte green alga Mantoniella squamata; the charophycean green algae Chara foetida, Coleochaete scutata, Klebsormidium flaccidum, and Mougeotia scalaris; the bryophytes Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica; and the lycopod Selaginella galleottii to get a better insight into the sequential evolution from green algae to land plants. The sequences were aligned with several previously published SSU rRNA sequences from chlorophytic and charophytic algae as well as from land plants to infer the evolutionary relationships for major evolutionary lineages within the Chlorobionta by distance matrix, maximum parsimony, and maximum likelihood analyses. Phylogenetic trees created by the different methods consistently placed the Charophyceae on the branch leading to the land plants. The Charophyceae were shown to be polyphyletic with the Charales (“charalean” algae) diverging earlier than the Coleochaetales, Klebsormidiales, Chlorokybales, and Zygnematales (“charophycean” algae) which branch from a point closer to the land plants in most analyses. Maximum parsimony and maximum likelihood analyses imply a successive evolution from “charophycean” algae, particularly Coleochaetales, to bryophytes, lycopods, and seed plants. In contrast, distance matrix methods group the bryophytes together with the “charophycean” algae, suggesting a separate evolution of these organisms compared with the club moss and the seed plants.

NEW CLASSIFICATION OF LIVERWORTS BASED ON MOLECULAR AND MORPHOLOGICAL DATA

Complete sequences for the 18S-rRNA gene of 22 bryophytes (12 completely new) were determined and used to construct phylogenetic trees. The evaluation of sequence data according to the maximum parsimony principle (PAUP 3.1.1) and the neighbor-joining method (MEGA) results in similar phylogenetic trees in which theBryopsida appear as a sister group to theJungermanniopsida, and both together as a sister group to theMarchantiopsida. Among theMarchantiopsida, theSphaerocarpales diverge early as a separate clade. TheMetzgeriales andJungermanniales are monophyletic. They belong to one clade and cannot be separated by either method of evaluation.

A Molecular Phylogeny of Bryophytes Based on the Nuclear Encoded 18S rRNA Genes

Summary Complete small ribosomal 18S RNA gene sequences from 6 liverworts, 6 mosses and 1 hornwort were used to reconstruct phylogenetic relationships between bryophytes. Sequence data were evaluated according to the maximum parsimony principle (PAUP 3.1.1) and the neighbour-joining method (MEGA). As outgroup, Chara foetida was used. For the first time molecular data support the open discussion about the relationship between Marchantiidae and Jungermanniidae liverworts, placing them regardless of the method used in different clades. The greatest absolute distances are found between Chara foetida and Marchantiidae followed by Sphagnum and Anthoceros. Chara foetida is closest to the Bryidae and Jungermanniidae.

Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: Basic data forHelianthus annuus (Asteraceae)

As a contribution for the study of systematic and evolutionary relationships it is suggested to analyze nuclear DNA and chromatin by means of CsCl ultracentrifugation, thermal denaturation and renaturation, scanning densitometry, and (ultra)structural analyses. Relevant data have been obtained forHelianthus annuus as a first example.The 2C DNA content of four cultivars ofHelianthus annuus L. was calibrated by comparative measurement withAllium cepa nuclei using a scanning densitometer in on-line operation with a computer. Significant infraspecific variation could be detected: cvar. “Amerikanische Riesen” displayed 6.1 pg, cvar. “Gefüllte Vielblütige” 9.9 pg, cvar. “Russian Mammoth” 8.9 pg, and a Heidelberg strain 8.7 pg.The buoyant density in neutral CsCl was determined for cvar. “Amerikanische Riesen” to be 1.695 g · cm−3; this corresponds to an average GC content of 35.1%. Thermal denaturation revealed a melting temperature of 86.4°C. Derivative thermal denaturation profiles led to the detection of several distinct DNA fractions.The species-specific nuclear structure is of the chromonematic type, but in differentiated cells the chromatin fibers may be more decondensed so that a chromomere-interchromomere structure appears. The heterochromatin constitutes an average of 4.5% of the total genome. Chromatin ultrastructure is characterized by a diffuse distribution of chromatin threads and patches. Nucleosomes of 110 Å diameter can be recognized.The data are discussed (a) in relation to findings on DNA variation in other plants, (b) in relation to the systematic usefulness and further characterization of nuclear DNA and chromatin, and (c) in relation to tissue-specific and functional variation of the species-specific chromatin structure.

Analysis of the ribosomal RNA gene repeat from the moss Funaria hygrometrica

Robosomal DNA from the moss Funaria hygrometrica was obtained by centrifugation of genomic DNA in a Hoechst 33258-CsCl density gradient. One of the three DNA-satellite bands (GC2) represented the ribosomal fraction. After digestion with EcoRI three larger fragments were obtained which hybridized to the large and small ribosomal subunits from Sinapis alba. The fragments were cloned and sequenced. The complete ribosomal DNA consists of 11 132 bp. The analysis of the 18S, 5.8S and 25S rDNA revealed a high sequence and length similarity to other plant sequences. An intron of 169 bp situated 10 bp from the 5′ end of the 25 S rDNA was found. The intergenic spacer (IGS) has a length of 5150 bp with 13 highly conserved subrepeats (38 bp except for two of 56 bp) followed directly by 6 other repeated elements of 42-43 bp. Two putative transcription initiation sites (TIS) were identified with some differences to known TIS of angiosperms.

Endopolyploidisierung wÄhrend des Streckungswachstums der Hypokotyle vonSinapis alba

SummaryThe relative DNA content (arbitrary units) per nucleus was determined in isolated nuclei of the etiolated hypocotyl ofSinapis alba by Feulgen microdensitometry and microfluorometry. An increase of endopolyploidy during elongation growth was detected. With increasing hypocotyl age (40 to 64 hours) the DNA concentration in the chromocenters increases. A hyperreplication of the DNA in the polyploid nuclei can be assumed. Biochemical determinations of DNA and the relative DNA amounts per nucleus determined by cytophotometry agree closely. Bacterial contamination has been excluded to account for the biochemically determined DNA increase.ZusammenfassungDurch Mikrodensitometrie und Mikröfluorometrie wird der DNA-Gehalt isolierter, feulgengefÄrbter Kerne aus etiolierten Hypocotylen vonSinapis alba bestimmt. Im Laufe des Streckungswachstums der Hypocotyle von 40–64 Stunden nehmen die endopolyploiden Zellen zu. Gleichzeitig steigt der DNA-Gehalt der Chromozentren stÄrker an, so da\ man eine überreplikation der DNA in den heterochromatischen Chromosomenabschnitten annehmen kann. Die biochemischen und die cytophotometrischen Bestimmungen der DNA-Menge stimmen gut überein. Eine Bakterienkontamination als Ursache für die biochemisch gemessene DNA-Zunahme kann auf Grund der direkten Kernmessungen ausgeschlossen werden.

Endopolyploidy inHelianthus annuus (Asteraceae), A scanning cytophotometric study

Endopolyploidy has been detected in some varieties ofHelianthus annuus L. (Asteraceae/Compositae) by means of scanning photometry of Feulgen-stained nuclei and analysis of nuclear structure. In the hypocotyl cells of seedlings, ploidy levels reach respectively 8 C and 16 C in different varieties, in the root cells 8 C and 16 C; in the cotyledons of ripening seeds 4 C to 8 C values have been found, while all nuclei of the inflorescence axis of one variety exhibit a DNA content of 4 C.—This is the first report of endopolyploidy in a non-succulentAsteraceae species. The characteristic distribution of the endopolyploidy levels in different varieties suggests a strong genetic and/or hormonal control of the final nuclear DNA content in differentiated cells.

Über die Synthese von DNS in den Keimpflanzen von Sinapis alba L.

SummarySeedlings of Sinapis alba were grown under standard conditions. In the hypocotyls and cotyledons DNA synthesis still takes place 36 h after sowing. This synthesis decreases in the following 24 h, but an incorporation of 3H-thymidine was found 108 h after sowing.Autoradiographic studies demonstrate the incorporation of 3H-thymidine into cell nuclei. While some nuclei are homogeneously labelled, in other nuclei the radioactivity appears preferentially or exclusively in the chromocenters.A transfer into the dark of plants previously grown in light (for 24 h or 48 h) does not result in an increase of DNA-synthesis again.Seedlings of Sinapis alba were grown under standard conditions. In the hypocotyls and cotyledons DNA synthesis still takes place 36 h after sowing. This synthesis decreases in the following 24 h, but an incorporation of (3)H-thymidine was found 108 h after sowing.Autoradiographic studies demonstrate the incorporation of (3)H-thymidine into cell nuclei. While some nuclei are homogeneously labelled, in other nuclei the radioactivity appears preferentially or exclusively in the chromocenters.A transfer into the dark of plants previously grown in light (for 24 h or 48 h) does not result in an increase of DNA-synthesis again.

Die lag-Phase bei der FUDR-Wirkung auf DNA-Synthese und Streckungswachstum bei Sinapis alba

In the seedlings of Sinapis alba, the lag-phase between the application of 5-FUDR and the beginning of the inhibition of elongation growth and the inhibition of DNA-synthesis has been studied. The elongation was retarded after 7 h, and then, depending on the concentration of the FUDR, was completely stopped. In the cotyledons the DNA-synthesis was strongly reduced after about 50 minutes, and in the hypocotyls a lag-phase of less than 30 minutes was observed. With the addition of thymidine the DNA-synthesis was immediately resumed, while the growth began with a lag-phase of 5-7 h. In every case the change in the DNA-synthesis preceded the change in the elongation growth. The inhibition of elongation growth could, therefore, be the consequence of inhibition of DNA-synthesis.SummaryIn the seedlings of Sinapis alba, the lag-phase between the application of 5-FUDR and the beginning of the inhibition of elongation growth and the inhibition of DNA-synthesis has been studied. The elongation was retarded after 7 h, and then, depending on the concentration of the FUDR, was completely stopped. In the cotyledons the DNA-synthesis was strongly reduced after about 50 minutes, and in the hypocotyls a lag-phase of less than 30 minutes was observed. With the addition of thymidine the DNA-synthesis was immediately resumed, while the growth began with a lag-phase of 5–7 h. In every case the change in the DNA-synthesis preceded the change in the elongation growth. The inhibition of elongation growth could, therefore, be the consequence of inhibition of DNA-synthesis.