Plant mitochondrial RNA editing.

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 (Charles). 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.

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

Ribosomal 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.

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.

Determination of 5-methylcytosine from plant DNA by high-performance liquid chromatography.

The relative amounts of the five nucleosides (deoxycytidine, 5-methyldeoxycytidine, deoxyadenosine, deoxyguanosine and thymidine) in the DNA of nine plant species, one plant satellite DNA, and one animal species were determined by high performance liquid chromatography. The method allows the clean separation of the nucleosides from 10 microgram samples with 15 min. The following values for the proportion of methylated cytosines among all cytosines were obtained: Lobularia maritima 18.5%, Nicotiana tabacum 32.6%, Pisum sativum 23.2%, Rhinanthus minor 29.2%, Sinapsis alba 12.2%, Vicia faba 30.5%, Viscum album 23.2%, Cymbidium pumilum 18.8%, Cymbidium pumilum AT-rich satellite DNA 15.8%, Triticum aestivum 22.4%. DNA of an animal, the gerbil, Meriones unguiculatus, had a methylation percentage of 3.1%. An estimate of the GC content based on the buoyant density of DNA tends to be lower than the actual value, an estimate based on the melting temperature tends to be higher. This supports the finding by other authors that DNA methylation decreases the buoyant density and may increase the melting temperature at high m5C concentration.

The mitotic Ca2+-adenosine triphosphatase in proliferating tissue of Vicia faba.

The mitotic Ca2+-ATPase was isolated from root tips of Vicia faba. The enzyme shows the same characteristics as the one isolated from a variety of animal cells. The results support the hypothesis that the membrane-bound mitotic Ca2+-ATPase is part of a Ca2+-regulating system universal for both plant and animal cells.

An A + T-rich satelitte DNA in a monocotyledonous plant, Cymbidium.

The DNA of aseptically grown protocorms of a Cymbidium hybrid and in vitro developed leaves, as well as DNA of leaves and flower buds of Cymbidium ceres from the greenhouse, was analysed by analytical ultracentrifugation and thermal denaturation. Upon ultracentrifugation a satellite DNA with a buoyant density of 1.682 g/cm-3 appears as a shoulder on the main band (density 1.694 g/cm-3). Thermal denaturation reveals an inhomogeneous main peak with the major component melting at 84 degrees C and a separate peak melting at 75 degrees C. This is the first demonstration of a satellite DNA in a monocot, and one of the rare examples of a major A + T-rich DNA fraction in a plant.

[Evidence of DNA-synthesis in hypocotyls and cytoledons of inhibited and non-inhibited seedlings of Sinapis alba]. / Nachweis der DNA-Synthese in Hypocotylen und Cotyledonen gehemmter und nicht gehemmter Keimlinge von Sinapis alba.

Etiolated elongating hypocotyls of Sinapis alba synthesize DNA 40-72 h after sowing. The increase in DNA content is about 62.5%. Four-hour pulse experiments showed an increasing rate of [(32)P]orthophosphate incorporation; the maximum rate was obtained 64-68 h after sowing. In the cotyledons DNA increase up to the 56th h after sowing; later the DNA-content remains constant. During this time the incorporation of (32)P into the DNA of the cotyledons decreases continuously.After simultaneous application of Fluordesoxyuridin (FdUrd) (4×10(-5)M) and thymidine (4×10(-4)M) or thymidylate (4×10(-4)M) 40-64 h after sowing, the growth rate was the same as that in H2O treated controls, whereas application of FdUrd (4×10(-5)M) alone inhibited growth.In the presence of FdUrd (4×10(-5)M) thymidine (4×10(-4)M) and thymidylate (4×10(-4)M) enhanced the specific activity of the DNA up to respectively 68% and 63% of that in the H2O treated controls. Hydroxyurea (4 mg/ml) inhibited DNA-synthesis and growth in the same manner as FdUrd.The prolonged DNA-synthesis and the reversal of the FdUrd effect by thymidine or thymidylate are discussed.

[The lag-phase of the effect of 5-FUDR on the DNA-synthesis and elongation growth in Sinapis alba]. / 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.

[Inhibition of Elongation of Hypocotyls of Sinapis alba with FUDR]. / Streckungshemmung durch FUDR bei den Hypokotylen von Sinapis alba.

Seedlings of Sinapis alba which were grown in light for 24, 48 and 72 h after 36 h of dark treatment renew elongation when transferred to dark again. The rate of elongation decreases with increased light treatment. The per cent as well as absolute inhibition of elongation by FUDR decreases with the duration of light treatment. The shortening of the hypocotyl is due mainly to the inhibition of cell elongation. The inhibition is not directly proportional to DNA synthesis at any particular time.Plants without cotyledons are less inhibited than those with cotyledons. Cytosine arabinoside is inhibitory only at high concentrations. According to these results elongation inhibition by FUDR does not involve the entire DNA-synthesis.

[DNA synthesis in the seedlings of Sinapis alba L]. / Über die Synthese von DNS in den Keimpflanzen von Sinapis alba L.

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.