Rainer M. Maier

Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I of the five distinguishable Euoenothera plastomes

Continued sequencing of Oenothera plastid chromosomes in our laboratory has led to the discovery of various errors in the published plastome I sequence (Hupfer et al. 2000). Some of these errors have also been noted by others [Rice DW, Palmer JD (2006) BMC Biol 4:31]. We have now re-sequenced the plastome I chromosome [Greiner S, Wang X, Rauwolf U, Silber MV, Mayer K, Meurer J, Harberer G, Herrmann RG (2008) The complete nucleotide sequences of the Wve genetically distinct plastid genomes of Oenothera subsection Oenothera: I. Sequence evaluation and plastome evolution. Nucleic Acids Res 36:2366–2378] and deposited the revised sequence in GenBank under the accession no. AJ271079.3. This submission supersedes the previously submitted sequences with the accession nos. AJ271079.1 and AJ271079.2. We wish to emphasize that the basic inferences drawn in the original publication (Hupfer et al. 2000) remain unchanged. We sincerely regret any inconvenience caused by the publication of the original data.

Disruption of plastid-encoded RNA polymerase genes in tobacco : expression of only a distinct set of genes is not based on selective transcription of the plastid chromosome

Abstract Plastids of higher plants operate with at least two distinct DNA-dependent RNA polymerases, which are encoded in the organelle (PEP) and in the nucleus (NEP), respectively. Plastid run-on assays and Northern analyses were employed to analyse gene expression in tobacco mutant plastids lacking the PEP genes rpoA, rpoB or rpoC1. Hybridisation of run-on transcripts to restriction fragments representing the entire tobacco plastid chromosome, as well as to selected plastid gene-specific probes, shows that all parts of the plastid DNA are transcribed in rpo-deficient plastids. In comparison to wild-type chloroplasts, which are characterized by preferential transcription of photosynthesis-related genes in the light, mutant plastids exhibit a different transcription pattern with less pronounced differences in the hybridisation intensities between the individual genes. The analysis of steady-state transcript patterns and transcription rates of selected genes in both types of plastids demonstrates that differences in transcription rates are not necessarily paralleled by corresponding changes in transcript levels. The accumulation of large transcripts in the mutant plastids indicates that processing of primary transcripts may be impaired in the absence of PEP. These data suggest that, contrary to the prevailing view, much of the regulation of NEP-driven plastid gene expression in the rpo-deficient mutants is not based on differential promoter usage but is exerted at post-transcriptional levels.

Pigment Deficiency in Nightshade/Tobacco Cybrids Is Caused by the Failure to Edit the Plastid ATPase α-Subunit mRNA

The subgenomes of the plant cell, the nuclear genome, the plastome, and the chondriome are known to interact through various types of coevolving macromolecules. The combination of the organellar genome from one species with the nuclear genome of another species often leads to plants with deleterious phenotypes, demonstrating that plant subgenomes coevolve. The molecular mechanisms behind this nuclear–organellar incompatibility have been elusive, even though the phenomenon is widespread and has been known for >70 years. Here, we show by direct and reverse genetic approaches that the albino phenotype of a flowering plant with the nuclear genome of Atropa belladonna (deadly nightshade) and the plastome of Nicotiana tabacum (tobacco) develops as a result of a defect in RNA editing of a tobacco-specific editing site in the plastid ATPase α-subunit transcript. A plastome-wide analysis of RNA editing in these cytoplasmic hybrids and in plants with a tobacco nucleus and nightshade chloroplasts revealed additional defects in the editing of species-specific editing sites, suggesting that differences in RNA editing patterns in general contribute to the pigment deficiencies observed in interspecific nuclear–plastidial incompatibilities.

Heterologous, splicing‐dependent RNA editing in chloroplasts: allotetraploidy provides trans‐factors

RNA editing is unique among post‐transcriptional processes in plastids, as it exhibits extraordinary phylogenetic dynamics leading to species‐specific editing site patterns. The evolutionary loss of a site is considered to entail the loss of the corresponding nuclear‐encoded site‐specific factor, which prevents the editing of foreign, i.e. heterologous, sites. We investigated the editing of short ‘spliced’ and ‘unspliced’ ndhA gene fragments from spinach in Nicotiana tabacum (tobacco) in vivo using biolistic transformation. Surprisingly, it turned out that the spinach site is edited in the heterologous nuclear background. Furthermore, only exon–exon fusions were edited, whereas intron‐containing messages remained unprocessed. A homologue of the spinach site was found to be present and edited in Nicotiana tomentosiformis, representing the paternal parent, but absent from Nicotiana sylvestris, representing the maternal parent of tobacco. Our data show that: (i) the cis‐determinants for ndhA editing are split by an intron; (ii) the editing capacity cannot be deduced from editing sites; and (iii) allopolyploidization can increase the editing capacity, which implies that it can influence speciation processes in evolution.

A promiscuous chloroplast DNA fragment is transcribed in plant mitochondria but the encoded RNA is not edited

The RNA editing processes in chloroplasts and mitochondria of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. AnrpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrialrpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potentialrpoB editing sites previously detected in maize were found to be edited in the rice chloroplastrpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events.

PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

Abstract. The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.

C-to-U conversion in the intercistronic ndhI/ndhG RNA of plastids from monocot plants: conventional editing in an unconventional small reading frame?

Abstract. Editing of plastid RNAs proceeds by C-to-U, in hornwort species also by extensive U-to-C, transitions, which predominantly lead to the restoration of codons for structurally and/or functionally important, conserved amino acid residues. So far, only one instance of editing outside coding regions has been reported – in the psbL/psbF intergenic region of Ginkgo biloba. This site was proposed to have no functional importance. Here we present an evaluation of an editing site in the ndhI/ndhG intergenic region in a related group of monocot plants. Efficient editing of this site, as well as the phylogenetic conservation of the resulting uridine residue, point at an important role for the sequence restored by editing. Two potential functions can be envisaged. (1) RNA secondary structure predictions suggest that the C-to-U conversion at this site can lead to a modified stem/loop structure of the ndhG 5′ UTR, which could influence ndhG expression. (2) Alternatively, editing of the ndhI/ndhG intergenic region may tag a so far unidentified small (12-codon) ORF, and lead to the restoration of a conserved phenylalanine codon. A screen with specific antibodies elicited against the putative peptide failed to detect such a peptide in chloroplast fractions. However, this failure may be attributable to its low and/or development-specific expression.

Is clustering of plastid RNA editing sites a consequence of transitory loss of gene function? – Implications for past environmental and evolutionary events in plants

Abstract Editing changes individual nucleotides in RNA thereby distinguishing the mature transcript sequence from the corresponding genomic sequence. Most editing events in chloroplasts change cytosine to uracil bases at specific positions in transcripts. In hornwort and fern species uracil to cytosine conversions also take place. RNA editing in plastids probably emerged among the first land plants and affects different genes of different plants in different positions. A significant clustering of editing sites in certain genes is found within related taxa. The clustering is particularly evident in ndh genes in angiosperms, where editing sites account for about 50% of total (near 30) editing sites of plastids and are rather similar in a large number of species. In gymnosperms, ndh genes are edited to a much lower extent, and the few editing sites identified in bryophytes are different from those of angiosperms. On the basis of these data and the role of ndh genes in the protection against photooxidative stress, we make the following hypothesis for the origin and evolution of editing sites: (i) primary mutations accumulated in genes which became dispensable for plants in certain ecosystems; (ii) under changing environments, the functionality of these genes was recovered in some of the descendants by transcript editing; and (iii) secondary mutations led to a subsequent and gradual disappearance of editing sites. Evidence and further tests for this hypothesis are discussed.