The commitment of eukaryotic cells to division normally occurs during the G1 phase of the cell cycle. In mammals D-type cyclins regulate the progression of cells through G1 and therefore are important for both proliferative and developmental controls. Plant CycDs (D-type cyclin homologs) have been identified, but their precise function during the plant cell cycle is unknown. We have isolated three tobacco (Nicotiana tabacum) CycD cyclin cDNAs: two belong to the CycD3 class (Nicta;CycD3;1 and Nicta;CycD3;2) and the third to the CycD2 class (Nicta;CycD2;1). To uncouple their cell-cycle regulation from developmental control, we have used the highly synchronizable tobacco cultivar Bright Yellow-2 in a cell-suspension culture to characterize changes in CycD transcript levels during the cell cycle. In cells re-entering the cell cycle from stationary phase, CycD3;2 was induced in G1 but subsequently remained at a constant level in synchronous cells. This expression pattern is consistent with a role for CycD3;2, similar to mammalian D-type cyclins. In contrast, CycD2;1 and CycD3;1 transcripts accumulated during mitosis in synchronous cells, a pattern of expression not normally associated with D-type cyclins. This could suggest a novel role for plant D-type cyclins during mitosis.

RNA editing has been documented in mitochondria of higher plants, notably dicots and monocots. To determine the distribution of mitochondrial RNA editing in the plant kingdom, we have now undertaken a survey of evolutionarily distant plants. RNA editing occurs in all major groups of land plants except the Bryophyta, suggesting that this process is an ancient trait that was established before the radiation of kormophyte plants. No editing is observed in representatives of the green algae, suggesting that editing arose in early land plants after the split of the Bryophyta or has been lost selectively in both algae and mosses. In ferns several U -* C changes are observed, one of which eliminates a genomically encoded UAA termination codon and creates a functional open reading frame.RNA editing has been observed in mitochondria and chloroplasts of angiosperm plants belonging to the Dicotyledonae and Monocotyledonae (1-3). In both organelles specific cytidines of the primary transcripts are altered to uridines in the mature mRNAs. Only a few instances of editing have been found in chloroplasts (4-6), whereas numerous editing events have been documented in mitochondria of higher plants for almost all mRNAs investigated (7-11). In the more thoroughly investigated higher plant species, all of the open reading frames encoding conserved functional polypeptides appear to be edited to some extent and the total number of editing sites documented amounts to 294 in wheat (11) and to >400 in Oenothera (10). The observed effect ofthis extensive editing on the encoded polypeptide sequences suggests that many of the higher plant mitochondrial genes would not encode functionally competent products without appropriate "correction" by RNA editing.Mitochondrial RNA editing has been reported in a number of angiosperm species, including the monocots wheat and maize and the dicots Oenothera, Sorghum, Petunia, Daucus, and Arabidopsis (7-11). The observation of analogous RNA editing in both monocots and dicots suggests that the origin of this process predates the divergence of the two lineages and is generally present in angiosperms. Recently, RNA editing has also been described in the gymnosperm Thuja (12). However, none of the plant species presumed to be closer to the evolutionary origin of flowering plants has been investigated. The only mitochondrial sequence data available allowing any inference about the need of RNA editing in nonflowering plants are from two bryophytes.Analysis of protein sequences deduced from the complete genomic nucleotide sequence of the liverwort Marchantia polymorpha (13) and comparison of several cDNA and genomic DNA sequences (14) did not show any evidence of RNA editing. Genomic sequence analysis of a mitochondrial gene in the moss Physcomitrella patens (15) allows a similar deduction, suggesting that RNA editing might not occur in mitochondria of the two bryophytes.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adve...

SummaryIn this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a speci®c mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the ®rst intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two-dimensional in organello mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or posttranslational steps in the synthesis of other mitochondrial proteins are affected. All of these defects cosegregated with the abnormal phenotype in the offspring of a NMS1 3 wild-type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.

SummaryProtein kinase CK2 is an ubiquitous Ser/Thr kinase essential for cell growth. We have used the highly synchronizable tobacco BY-2 cell line to investigate whether CK2 activity and expression are regulated in a cell cycle phase-dependent manner in higher plants. Speci®c cDNA probes for tobacco CK2a and b subunits, respectively, and polyclonal antibodies recognising a and b subunits separately, were obtained to determine mRNA and protein levels of both subunits. Our results show that CK2 activity oscillates throughout the cell cycle, peaking at G1/S and M phases, due to a posttranslational regulation of the tetrameric enzyme. Additional levels of control of CK2 expression operate in relation to the proliferative state of the cells, including differential accumulation of a and b transcripts and posttranscriptional regulation of protein levels (b subunit). Moreover, in vivo inhibition of CK2 activity corroborates the requirement of the functional CK2 to progress through the cell division cycle, and suggests that CK2 might play an important role at the G2/M checkpoint.

We previously reported that the Nicotiana sylvestris CMSII mutant mitochondrial DNA carried a large deletion. Several expressed sequences, most of which are duplicated, and the unique copy of the nad7 gene encoding the NAD7 subunit of the NADH:ubiquinone oxidoreductase complex (complex I) are found in the deletion. Here, we show that the orf87‐nad3‐nad1/A cotranscription unit transcribed from a unique promoter element in the wild‐type, is disrupted in CMSII. Nad3, orf87 and the promoter element are part of the deleted sequence, whilst the nad1/A sequence is present and transcribed from a new promoter brought by the recombination event, as indicated by Northern and primer extension experiments. However, Western analyses of mitochondrial protein fractions and of complex I purified using anti‐NAD9 affinity columns, revealed that NAD1 is lacking in CMSII mitochondria. Our results suggest that translation of nad1 transcripts rather than transcription itself could be altered in the mutant. Consequences of lack of this submit belonging the membrane arm of complex I and thought to contain the ubiquinone‐binding site, are discussed.