Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation.

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns. MatK is essential for plant cell survival in tobacco, and thus null mutants have not yet been isolated. We therefore attempted to over-express MatK from a neutral site in the chloroplast, placing it under the control of a theophylline-inducible riboswitch. This ectopic insertion of MatK lead to a variegated cotyledons phenotype. The addition of the inducer theophylline exacerbated the phenotype in a concentration-dependent manner. The extent of variegation was further modulated by light, sucrose and spectinomycin, suggesting that the function of MatK is intertwined with photosynthesis and plastid translation. Inhibiting translation in the transplastomic lines has a profound effect on the accumulation of several chloroplast mRNAs, including the accumulation of an RNA antisense to rpl33, a gene coding for an essential chloroplast ribosomal protein. Our study further supports the idea that MatK expression needs to be tightly regulated to prevent detrimental effects and establishes another link between leaf variegation and chloroplast translation.

In vitro promoter recognition by the catalytic subunit of plant phage-type RNA polymerases.

KEY MESSAGE: We identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm (mitochondrial RNAP), RPOTp (plastidial RNAP), and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a 'specificity loop'. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed. The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm-the mitochondrial RNAP, RPOTp-the plastidial RNAP, and RPOTmp-an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.

Mitochondrial atp9 genes from petaloid male-sterile and male-fertile carrots differ in their status of heteroplasmy, recombination involvement, post-transcriptional processing as well as accumulation of RNA and protein product.

KEY MESSAGE: Petaloid cytoplasmic male-sterile carrots exhibit overexpression of the mitochondrial atp9 genes which is associated with specific features in organization and expression of these sequences. In carrots, the Sp-cytoplasm causes transformation of stamens into petal-like organs, while plants carrying normal N-cytoplasm exhibit normal flower morphology. Our work was aimed at characterization of distinct features both cytoplasms display with respect to organization and expression of the mitochondrial atp9 genes. We show that two carrot atp9 genes, previously reported as cytoplasm-specific, in fact occur in heteroplasmic condition. In the Sp-cytoplasm the atp9-1 version dominates over atp9-3, while in N-cytoplasmic plants this proportion is reversed. Herein, we also indicate the presence and recombination activity of a 130-/172-bp sequence repeat which likely shaped the present organization of carrot atp9 loci. Furthermore, cDNA sequence examination revealed that the atp9 open reading frames (ORFs) were C to U edited in 4 nucleotide positions. One of the editing events turns a glutamine triplet into the stop codon, thereby equalizing ORFs of atp9-1 and atp9-3. A certain fraction of partially edited molecules was identified-they all represented the atp9-3 sequence. In either Sp- or N-cytoplasmic plants multiple 5' transcript termini were observed. Of these, the ones mapping more distantly from the atp9 ORF were more pronounced in case of petaloid accessions. It was also shown that despite comparable copy number of the genomic atp9 sequences, the level of the respective mRNAs was approximately 3 times higher in case of petaloid carrots. The latter fact corresponded to the elevated content of the ATP9 protein in plants carrying Sp-cytoplasm. The semi-fertile phenotype of such plants is associated with a drop in ATP9 accumulation.

Quantification of organellar DNA and RNA using real-time PCR.

Quantitative (real-time) polymerase chain reaction (PCR) allows the measurement of relative organellar gene copy numbers as well as transcript abundance of individual mitochondrial or plastidial genes. Requiring only minute amounts of total DNA or RNA, the described method can replace traditional analyses like Southern or Northern hybridization which require large amounts of organellar nucleic acids and usually provide only semiquantitative data. Here we describe prerequisites, reaction conditions, and data analysis principles, which should be applicable for a wide range of plant species and experimental situations where comparative and precise determination of gene copy numbers or transcript abundance is requested. Sequences of amplification primers for qPCR of organellar genes from Arabidopsis are provided.

Chloroplast DNA in mature and senescing leaves: a reappraisal.

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed high-resolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ∼30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.

A third mitochondrial RNA polymerase in the moss Physcomitrella patens.

In most organisms, the mitochondrial genes are transcribed by RNA polymerases related to the single-subunit RNA polymerases of bacteriophages like T3 and T7. In flowering plants, duplication(s) of the RpoTm gene coding for the mitochondrial RNA polymerase (RPOTm) led to the evolution of additional RNA polymerases transcribing genes in plastids (RPOTp) or in both mitochondria and plastids (RPOTmp). Two putative RPOTmp enzymes were previously described to be encoded by the nuclear genes RpoTmp1 and RpoTmp2 in the moss Physcomitrella patens. Here, we report on a third Physcomitrella RpoT gene. We determined the sequence of the cDNA. Comparison of the deduced amino acid sequence with sequences of plant organellar RNA polymerases suggests that this gene encodes a functional phage-type RNA polymerase. The 78 N-terminal amino acids of the putative RNA polymerase were fused to GFP and found to target the fusion protein exclusively to mitochondria in Arabidopsis protoplasts. P. patens is the only known organism to possess three mitochondrial RNA polymerases.

Light-dependent, plastome-wide association of the plastid-encoded RNA polymerase with chloroplast DNA.

Plastid genes are transcribed by two types of RNA polymerases: a plastid-encoded eubacterial-type RNA polymerase (PEP) and nuclear-encoded phage-type RNA polymerases (NEPs). To investigate the spatio-temporal expression of PEP, we tagged its α-subunit with a hemagglutinin epitope (HA). Transplastomic tobacco plants were generated and analyzed for the distribution of the tagged polymerase in plastid sub-fractions, and associated genes were identified under various light conditions. RpoA:HA was detected as early as the 3rd day after imbibition, and was constitutively expressed in green tissue over 60 days of plant development. We found that the tagged polymerase subunit preferentially associated with the plastid membranes, and was less abundant in the soluble stroma fraction. Attachment of RpoA:HA to the membrane fraction during early seedling development was independent of DNA, but at later stages of development, DNA appears to facilitate attachment of the polymerase to membranes. To survey PEP-dependent transcription units, we probed for nucleic acids enriched in RpoA:HA precipitates using a tobacco chloroplast whole-genome tiling array. The most strongly co-enriched DNA fragments represent photosynthesis genes (e.g. psbA, psbC, psbD and rbcL), whose expression is known to be driven by PEP promoters, while NEP-dependent genes were less abundant in RpoA:HA precipitates. Additionally, we demonstrate that the association of PEP with photosynthesis-related genes was reduced during the dark period, indicating that plastome-wide PEP-DNA association is a light-dependent process.

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

The transcription machineries of plant mitochondria and chloroplasts: Composition, function, and regulation.

Although genomes of mitochondria and plastids are very small compared to those of their bacterial ancestors, the transcription machineries of these organelles are of surprising complexity. With respect to the number of different RNA polymerases per organelle, the extremes are represented on one hand by chloroplasts of eudicots which use one bacterial-type RNA polymerase and two phage-type RNA polymerases to transcribe their genes, and on the other hand by Physcomitrella possessing three mitochondrial RNA polymerases of the phage type. Transcription of genes/operons is often driven by multiple promoters in both organelles. This review describes the principle components of the transcription machineries (RNA polymerases, transcription factors, promoters) and the division of labor between the different RNA polymerases. While regulation of transcription in mitochondria seems to be only of limited importance, the plastid genes of higher plants respond to exogenous and endogenous cues rather individually by altering their transcriptional activities.

Evolution of plant phage-type RNA polymerases: the genome of the basal angiosperm Nuphar advena encodes two mitochondrial and one plastid phage-type RNA polymerases.

BACKGROUND: In mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena. RESULTS: By screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP) exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp gene, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. CONCLUSIONS: Nuphar advena encodes two mitochondrial and one plastid phage-type RNAP. Identification of a plastid-localized phage-type RNAP in this basal angiosperm, orthologous to all other RpoTp enzymes of flowering plants, suggests that the duplication event giving rise to a nuclear gene-encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastididal RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that its occurrence is an evolutionary novelty of eudicotyledonous plants like Arabidopsis.

A mitochondrial rRNA dimethyladenosine methyltransferase in Arabidopsis.

S-adenosyl-L-methionine-dependent rRNA dimethylases mediate the methylation of two conserved adenosines near the 3' end of the rRNA in the small ribosomal subunits of bacteria, archaea and eukaryotes. Proteins related to this family of dimethylases play an essential role as transcription factors (mtTFBs) in fungal and animal mitochondria. Human mitochondrial rRNA is methylated and human mitochondria contain two related mtTFBs, one proposed to act as rRNA dimethylase, the other as transcription factor. The nuclear genome of Arabidopsis thaliana encodes three dimethylase/mtTFB-like proteins, one of which, Dim1B, is shown here to be imported into mitochondria. Transcription initiation by mitochondrial RNA polymerases appears not to be stimulated by Dim1B in vitro. In line with this finding, phylogenetic analyses revealed Dim1B to be more closely related to a group of eukaryotic non-mitochondrial rRNA dimethylases (Dim1s) than to fungal and animal mtTFBs. We found that Dim1B was capable of substituting the E. coli rRNA dimethylase activity of KsgA. Moreover, we observed methylation of the conserved adenines in the 18S rRNA of Arabidopsis mitochondria; this modification was not detectable in a mutant lacking Dim1B. These data provide evidence: (i) for rRNA methylation in Arabidopsis mitochondria; and (ii) that Dim1B is the enzyme catalyzing this process.

Biparental inheritance of plastidial and mitochondrial DNA and hybrid variegation in Pelargonium.

Plastidial (pt) and mitochondrial (mt) genes usually show maternal inheritance. Non-Mendelian, biparental inheritance of plastids was first described by Baur (Z Indukt Abstamm Vererbungslehre 1:330-351, 1909) for crosses between Pelargonium cultivars. We have analyzed the inheritance of pt and mtDNA by examining the progeny from reciprocal crosses of Pelargonium zonale and P. inquinans using nucleotide sequence polymorphisms of selected pt and mt genes. Sequence analysis of the progeny revealed biparental inheritance of both pt and mtDNA. Hybrid plants exhibited variegation: our data demonstrate that the inquinans chloroplasts, but not the zonale chloroplasts bleach out, presumably due to incompatibility of the former with the hybrid nuclear genome. Different distribution of maternal and paternal sequences could be observed in different sectors of the same leaf, in different leaves of the same plant, and in different plants indicating random segregation and sorting-out of maternal and paternal plastids and mitochondria in the hybrids. The substantial transmission of both maternal and paternal mitochondria to the progeny turns Pelargonium into a particular interesting subject for studies on the inheritance, segregation and recombination of mt genes.

Evolution of phage-type RNA polymerases in higher plants: characterization of the single phage-type RNA polymerase gene from Selaginella moellendorffii.

Selaginella moellendorfii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases (RpoTs) were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3006 nt, encoding a putative protein of 1002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. In phylogenetic analyses, the Selaginella RpoT polymerase is in a sister position to all other phage-type polymerases of angiosperms. However, according to its conserved exon-intron structure, the Selaginella RpoT gene is representative of the molecular evolutionary lineage giving rise to the RpoT gene family of flowering plants. The N-terminal transit peptide of SmRpoT is shown to confer targeting of green fluorescent protein exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. Angiosperms and the moss P. patens possess small gene families encoding RpoTs, which include mitochondrial- and chloroplast-targeted RNA polymerases. In striking contrast, the Selaginella RpoT gene is shown to be single-copy, although Selaginella, as a lycophyte, has a phylogenetic position between Physcomitrella and angiosperms. Thus, there is no evidence that Selaginella may contain a nuclear-encoded phage-type chloroplast RNA polymerase.

Arabidopsis phage-type RNA polymerases: accurate in vitro transcription of organellar genes.

The T7 bacteriophage RNA polymerase (RNAP) performs all steps of transcription, including promoter recognition, initiation, and elongation as a single-polypeptide enzyme. Arabidopsis thaliana possesses three nuclear-encoded T7 phage-type RNAPs that localize to mitochondria (RpoTm), plastids (RpoTp), or presumably both organelles (RpoTmp). Their specific functions are as yet unresolved. We have established an in vitro transcription system to examine the abilities of the three Arabidopsis phage-type RNAPs to synthesize RNA and to recognize organellar promoters. All three RpoT genes were shown to encode transcriptionally active RNAPs. RpoTmp displayed no significant promoter specificity, whereas RpoTm and RpoTp were able to accurately initiate transcription from overlapping subsets of mitochondrial and plastidial promoters without the aid of protein cofactors. Our study strongly suggests RpoTm to be the enzyme that transcribes most, if not all, mitochondrial genes in Arabidopsis. Intrinsic promoter specificity, a feature that RpoTm and RpoTp share with the T7 RNAP, appears to have been conserved over the long period of evolution of nuclear-encoded mitochondrial and plastidial RNAPs. Selective promoter recognition by the Arabidopsis phage-type RNAPs in vitro implies that auxiliary factors are required for efficient initiation of transcription in vivo.

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription.

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.

Development- and tissue-specific expression of the RpoT gene family of Arabidopsis encoding mitochondrial and plastid RNA polymerases.

Arabidopsis thaliana possesses three RpoT genes which encode three different phage-type RNA polymerases with yet unknown function in organelle transcription: RpoTm and RpoTp, imported into mitochondria and plastids, respectively, and RpoTmp, co-targeted into both organelles. Expression of the RpoT genes was analyzed by quantitative RT-PCR, histochemical beta-glucuronidase (GUS) assays and in situ hybridization. Transcripts of all three RpoT genes accumulated to very low amounts in all organs. Surprisingly, RT-PCR revealed their highest levels in flower tissues. RpoTm transcripts were the most abundant in all organs, except mature leaves, in which RpoTp transcripts showed the highest accumulation. In the developing seedling, RpoTm::GUS and RpoTmp::GUS expression precedes that of RpoTp::GUS, the latter showing up only 7 days after germination. The RpoTm and RpoTmp promoters expressed GUS mainly in meristematic and mitochondria-rich cells such as the distal part of the root and companion cells flanking the phloem, whereas RpoTp::GUS activity was found in green tissues as the parenchyme cells of young leaves, the primary cortex of the stem, and sepals of buds and young flowers. Sites of GUS expression coincided spatially with those of in situ hybridization. Our data demonstrate an overlapping expression pattern of RpoTm and RpoTmp, and a completely differing pattern of RpoTp expression. The results suggest that RpoTm and RpoTmp recognize different types of mitochondrial promoters. The plastid polymerase RpoTp might play a major role in green tissue, i.e. in chloroplast transcription, whilst the dual-targeted RpoTmp in plastids should function mainly in the transcription of genes in non-green types.

Multiple promoters are a common feature of mitochondrial genes in Arabidopsis.

Mitochondrial genes in the plant Arabidopsis thaliana are transcribed by two phage-type RNA polymerases encoded in the nucleus. Little is known about cis-elements that are recognized by these enzymes and mediate the transcription of the Arabidopsis mitochondrial genome. Here, 30 transcription initiation sites of 12 mitochondrial genes and gene clusters have been determined using 5'-RACE and ribonuclease protection analysis of primary transcripts labelled in vitro by guanylyltransferase. A total of 9 out of 12 genes were found to possess multiple promoters, revealing for the first time that multiple promoters are a common feature of mitochondrial genes in a dicotyledonous plant. No differences in promoter utilization were observed between leaves and flowers, suggesting that promoter multiplicity reflects a relaxed promoter specificity rather than a regulatory role of promoter selection. Nearly half the identified transcription initiation sites displayed immediately upstream a CRTA core sequence, which was mostly seen within the previously described CRTAAGAGA promoter motif or a novel CGTATATAA promoter element. About as many promoters possessed an ATTA or RGTA core. Our data indicate that the majority of mitochondrial promoters in Arabidopsis deviate significantly from the nonanucleotide consensus derived earlier for dicot mitochondrial promoters.

Chloroplast development affects expression of phage-type RNA polymerases in barley leaves.

We have identified the barley gene and cDNA encoding the plastid phage-type RNA polymerase (RNAP), nuclear-encoded plastid RNAP (RpoTp), and the nearly full-length cDNA of the mitochondrial RNAP, nuclear-encoded mitochondrial RNAP (RpoTm). RpoTp spans more than 9000 nt, consists of 19 exons and 18 introns, gives rise to a 3632-nt mRNA and is localized to the long arm of chromosome 1 (7H). The length of the deduced polypeptide is 948 residues. The mRNA levels of RpoTp and RpoTm were determined in roots and primary leaf sections of 7-day-old barley seedlings of the albostrians mutant, which were either phenotypically normal and exhibited a gradient of chloroplast development, or contained ribosome-deficient undifferentiated plastids. Transcript levels of RpoTp and RpoTm in almost all sections reached higher concentrations in plastid ribosome-deficient leaves than in the wild-type material, except in the most basal part of the leaf. These data indicate a role of plastid-to-nucleus signalling in the expression of the two RpoT genes. The mRNA levels of the plastid genes, beta-subunit of plastid-encoded RNAP (rpoB), proteolytic subunit of the Clp protease (clpP) and ribosomal protein Rpl2 (rpl2), all known to be transcribed by the nuclear-encoded RNAP (NEP), followed closely the pattern of RpoTp mRNA accumulation, strongly suggesting that RpoTp and NEP are identical. Transcripts of RpoTm and RpoTm-transcribed mitochondrial genes cytochrome oxidase subunit 2 (coxII) and ATPase subunit 9 (atp9) accumulated to the highest levels in the most basal parts of the leaf and declined considerably towards the leaf tip with a pronounced reduction in green versus white leaves. Our data revealed a marked influence of the developmental stage of the plastid on the expression and activity of organellar phage-type RNAPs and their target genes. Thus, interorganellar cross-talk in the regulated expression of nuclear-encoded plastid and mitochondrial RNAP genes might be a key element governing the concerted expression of genes located within plastids, mitochondria and the nucleus of the plant cell.

The C-terminal region of mitochondrial single-subunit RNA polymerases contains species-specific determinants for maintenance of intact mitochondrial genomes.

Functional conservation of mitochondrial RNA polymerases was investigated in vivo by heterologous complementation studies in yeast. It turned out that neither the full-length mitochondrial RNA polymerase of Arabidopsis thaliana, nor a set of chimeric fusion constructs from plant and yeast RNA polymerases can substitute for the yeast mitochondrial core enzyme Rpo41p when expressed in Deltarpo41 yeast mutants. Mitochondria from mutant cells, expressing the heterologous mitochondrial RNA polymerases, were devoid of any mitochondrial genomes. One important exception was observed when the carboxyl-terminal domain of Rpo41p was exchanged with its plant counterpart. Although this fusion protein could not restore respiratory function, stable maintenance of mitochondrial petite genomes (rho(-))(-) was supported. A carboxyl-terminally truncated Rpo41p exhibited a comparable activity, in spite of the fact that it was found to be transcriptionally inactive. Finally, we tested the carboxyl-terminal domain for complementation in trans. For this purpose the last 377 amino acid residues of yeast mitochondrial Rpo41p were fused to its mitochondrial import sequence. Coexpression of this fusion protein with C-terminally truncated Rpo41p complemented the Deltarpo41 defect. These data reveal the importance of the carboxyl-terminal extension of Rpo41p for stable maintenance of intact mitochondrial genomes and for distinct species-specific intramolecular protein-protein interactions.

Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids.

Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.