Tis

Plastid bacterial-type RNA polymerase (PEP)

RpoTp

Fig. 2. Schematic representation of RpoT phylogeny. Distances are proportional to relative sequence divergence; numbers at nodes show branch support values. RpoT gene duplications occurred several times during the evolution of land plants. Reconstruction of phylogeny was done by quartet puzzling (based on data published in Emanuel et al. 2006).

RpoTp

RpoT1,2

Physcomitrella

RpoTp

Monocots

Dicots Monocots

Fig. 2. Schematic representation of RpoT phylogeny. Distances are proportional to relative sequence divergence; numbers at nodes show branch support values. RpoT gene duplications occurred several times during the evolution of land plants. Reconstruction of phylogeny was done by quartet puzzling (based on data published in Emanuel et al. 2006).

observed an RNA polymerase activity of a 110 kDa protein (the size expected for RpoT products) that was prepared from spinach chloroplasts and initiated transcription from a T7 promoter but not from an rbcL PEP promoter. It has been known for many years that the mitochondrial genes of baker's yeast, Saccharomy-ces cerevisiae, are transcribed by a nuclear encoded phage-type RNA polymerase (Masters et al. 1987). It is now evident that related phage-type polymerases are responsible for mitochondrial transcription in nearly all eukaryotes. The only exceptions from this rule are freshwater protozoa like Reclinomonas belonging to the jakobids. These lower eukaryotes still possess genes for a bacterial-type RNA polymerase in their mitochondrial genomes which became lost during the evolution of this organelle in the other lineages of eukaryotes (Lang et al. 1997). Genes potentially coding for RNA polymerases of the phage-type are also found on so-called 'linear plasmids', double-stranded DNAs of around 10 kb that have been detected in the mitochondria of several protozoa, fungi, and plants. Neither the origin of these 'plasmids', that exhibit features of viral genomes, nor the functional roles of their genes are known (Meinhardt et al. 1997). Phylogenetic trees of phage-type RNA polymerases suggest that the nuclear gene for the mitochondrial RNA polymerase evolved independently of the plasmid-localized RNA poly-merase genes probably from an ancestral bacteriophage gene (Lysenko and Kuznetsov 2005; Azevedo et al. 2006; Emanuel et al. 2006). Genes coding for phage-type RNA polymerases duplicated several times during the evolution of plants (Fig. 2). They were first discovered in Arabidopsis and Chenopodium and designated as RpoT genes (for RNA polymerase of the phage T3/T7 type; Hedtke et al. 1997; Weihe et al. 1997). Meanwhile it is evident that the nuclear genomes of dicotyledonous and monocotyledonous plants contain more than one RpoT gene (Fig. 3). The diploid genomes of the eudicots Arabidopsis (Hedtke et al. 1997, 2000), Nicotiana silvestris (Kobayashi et al. 2001a, 2001b, 2002), and Populus (deduced from the sequences data in http://genome.jgi-psf.org; Tuskan et al. 2006) contain 3 RpoT genes. The amphidiploid genome of tobacco, N. tabacum, contains 6 RpoT genes (two sets of three genes, one set each from the two diploid parental species; Hedtke et al. 2002).

The N-termini of the different RpoT polymerases (RpoTm, RpoTp, and RpoTmp) fused to GFP (green fluorescence protein) target the protein to mitochondria, plastids, and both organelles, respectively (Hedtke et al. 1997, 1999, 2000, 2002; Kobayashi et al. 2001a, 2001b, 2002). It has therefore been suggested that the RpoT genes encode mitochondrial (RpoTm; the Arabidopsis gene was originally designated as RpoY and RpoT;1; Hedtke et al. 1997, 2000), plastid (RpoTp; originally RpoZ and RpoT;3), and dual-targeted RNA polymerases (RpoTmp; originally RpoT;2). Targeting to one or the other organelle might be regulated at the level of translation as the RpoTmp mRNAs contain two potential start codons for translation, a feature which is conserved for all RpoTmp messengers of dicots and even Physcomitrella (see below). If the first start codon with a position more close to the 5' end is used, a transit peptide is synthesized that allowed for transportation of the protein into both organelles. If the exclusive usage of the second start codon was forced by deletion of the first one, the smaller transit peptide imported GFP only into mitochondria (Hedtke et al. 2000, 2002; Kobayashi et al. 2001a; Richter et al. 2002).

Monocots (only cereals have so far been investigated) have only two RpoT genes, one coding for a mitochondrial (RpoTm), the other for a plastidial RNA polymerase (RpoTp; Chang et al. 1999; Ikeda and Gray 1999; Emanuel et al. 2004; Kusumi et al. 2004; Fig. 3). Also the moss Physcomitrella patens contains two RpoT genes (Kabeya et al. 2002; Richter et al. 2002). Other plants have not been studied so far. The Physcomitrella genes were named RpoTmp1 and RpoTmp2 by Richter et al. (2002), since it was observed that the putative transit peptides encoded by both genes mediated dual targeting of GFP to plastids and mitochondria like in the case of the RpoTmp polymerases of dicots. However, RpoTmp localization is still a matter of debate. Targeting of GFP to mitochondria but not to plastids was observed in Arabidopsis or Physcomitrella when the protein was fused not only with the putative RpoTmp targeting sequence but also with the 5-flanking UTR. For yet unknown reasons, the presence of the 5'-UTR prevents usage of the first start codon during translation of the Arabidopsis and Physcomi-trella RpoTmp mRNAs. As mentioned above, translation from the second start codon produces a transit peptide for import into mitochondria (Kabeya and Sato 2005). The authors proposed, therefore, that dual targeting of GFP fused to the putative RpoTmp transit peptide alone, i.e. without the 5-UTR, was an experimental artifact and these genes encode mitochondrial RNA polymerases. On the other hand, exclusive targeting to mitochondria is not in agreement with the observation

Fig. 3. Chlamydomonas possesses only one nuclear RpoT gene that is proposed to encode the mitochondrial RNA polymerase (mtRNAP). Cereals have two RpoT genes, one encoding the mtRNAP, the other a plastid RNA polymerase supposed to represent NEP, whereas Arabidopsis and other eudicots additionally acquired RpoTmp, which may contribute to transcription in mitochondria and plastids.

Fig. 3. Chlamydomonas possesses only one nuclear RpoT gene that is proposed to encode the mitochondrial RNA polymerase (mtRNAP). Cereals have two RpoT genes, one encoding the mtRNAP, the other a plastid RNA polymerase supposed to represent NEP, whereas Arabidopsis and other eudicots additionally acquired RpoTmp, which may contribute to transcription in mitochondria and plastids.

that the RpoTmp homolog of spinach was detected in chloroplasts but not in mitochondria with antibodies reacting specifically with this enzyme (Azevedo et al. 2006). Moreover, studies on mutants lacking RpoTmp suggested a function in plastid transcription (Baba et al. 2004; Hricova et al. 2006; see below). Obviously, more detailed investigations into the localization of RpoTmp are required and the possibility of a regulation of targeting to mitochondria and/or plastids at the level of translation should be considered (Christensen et al. 2005).

Whether algae need NEP in addition to PEP to transcribe their plastid genes is not known yet (see review by Smith and Purton 2002). Like higher plants, algae bear genes for the core subunits of PEP in their plastid genomes. In contrast to Epifagus (see above), even the nonphotosynthetic alga Astasia longa, the malaria parasite Plasmodium falciparum, and related organisms have plastid (apicoplast) PEP genes (Wilson et al. 1996; Gockel and Hachtel 2000; Sheveleva et al. 2002) suggesting that a NEP activity is lacking. The nuclear genome of Chlamydomonas contains only one RpoT gene (A. Weihe et al., unpublished data). There are no experimental data on the subcellular localization of the Chlamydomonas RpoT gene product, but it likely codes for the mitochondrial RNA polymerase as in the other eukaryotes that possess only a single gene for a phage-type RNA polymerase (Fig. 3). Inhibition of transcription in Chlamydomonas chloroplasts by inhibitors which are specific for the bacterial-type RNA polymerase and would not affect the activity of phage-type RNA polymerases led to a complete block of plastid gene expression arguing against the presence of NEP activity in this alga (Surzycki 1969; Guertin and Bellemare 1979). Also another alga, Osteococcus tauri, possesses only one RpoT gene, which likely encodes the mitochondrial RNA polymerase (W. Hess, T. Börner, H. Moreau, unpublished; Derelle et al. 2006).

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