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
© Yin et al; licensee BioMed Central Ltd. 2010
Received: 20 July 2010
Accepted: 6 December 2010
Published: 6 December 2010
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.
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.
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.
In the mitochondria of all eukaryotes, with the exception of jacobids, the bacterial-type RNA polymerase of the former endosymbiont has been replaced by a T-odd phage-type RNA polymerase (for review, see ). The mitochondrial genome of the jacobid Reclinomonas americana encodes a bacterial-type RNAP [2, 3], whose expression has still to be demonstrated. Likewise, chloroplast genomes have retained the rpoA, B, and C genes of their cyanobacterial ancestor, which encode the core subunits of the plastid-encoded plastid RNAP (PEP). Additionally, mono- and eudicotyledonous plants were found to require a second, nuclear gene-encoded plastid RNAP activity (NEP) to transcribe their chloroplast genes [1, 4, 5]. Phage-type RNA polymerases were identified as representing this NEP activity [6–8]. Thus, in mono- and eudicots, nuclear gene-encoded phage-type RNA polymerases (RpoT polymerases) not only transcribe the mitochondrial genome but are also involved in the transcription of the plastid genome [1, 5, 9]. Genes encoding phage-type RNA polymerases have been identified in the nuclear genomes of various flowering plants, like Chenopodium album , Arabidopsis thaliana [7, 11], Nicotiana ssp. [12–14], Zea mays , wheat , barley , and rice . The moss Physcomitrella patens contains three RpoT genes [19, 20], genome project data, http://www.phytozome.net/physcomitrella. Two of the Physcomitrella RpoTs are potentially capable of being targeted to both mitochondria and chloroplasts , whereas the third gene encodes an RNAP of exclusively mitochondrial localization (U. Richter, unpublished data). Eudicots like Arabidopsis and Nicotiana harbor three phage-type RNA polymerases as well, but their localization within the cell differs from the Physcomitrella enzymes. Eudicots possess a mitochondrial (RpoTm), a plastid (RpoTp) and a dual-targeted phage-type RNA polymerase (RpoTmp; [11, 13, 14]), the latter involved in the transcription of mitochondrial and plastid genes [21–24]. No phage-type NEP has been detected in algae thus far. In Chlamydomonas, only one RpoT gene was identified (Weihe et al., unpublished data; genome project data, http://genome.jgi-psf.org/Chlre4/Chlre4.home.html), presumably encoding a mitochondrial-localized RNAP. The single-copy RpoT genes identified in the genomes of other green algae (Ostreococcus, Micromonas), most likely, encode mitochondrial RNA polymerases. Multiple phage-type RNA polymerases are only found in land plant species. Maier and colleagues  proposed that this feature could either be a prerequisite for the spatio-temporal regulatory needs of embryophytes and an adaption to the peculiar requirements of a terrestrial life style or it might be the mere result of the specifics of the plant organelle genetic systems in interaction with the nuclear genome (transgenomic suppression of point mutations). In this context it is interesting to note that the lycophyte Selaginella moellendorffii possesses also only a single RpoT polymerase, which likely is exclusively active in mitochondria . Thus, there seems to be no NEP activity in the lycophytes. Like the Physcomitrella RpoTs, the Selaginella polymerase is separated in phylogentic trees from the angiosperm clade, which forms two groups: plastid-localized enzymes on one hand, and mitochondrial and dual-targeted polymerases on the other [1, 5]. The origin of the NEP activity as found in mono- and eudicots and of the dual-targeted RpoT polymerases observed in eudicots remains unclear.
To gain a deeper insight into the evolution of phage-type RNA polymerases in the plant lineage and to deepen our understanding of the significance of multiple phage-type RNAP activities in both mitochondria and plastids we have investigated the waterlily Nuphar advena. Together with Amborella, Liriodendron and Acorus, Nuphar is one of the most studied basal angiosperms. As one of the deepest branching angiosperms, Nuphar has become an important model plant for understanding the origin of key angiosperm innovations. Here, we report the identification and characterization of three RpoT genes from Nuphar advena. Our data indicate that Nuphar advena (and possibly other basal angiosperms) possesses two mitochondrial-localized phage-type RNAPs as well as already a plastid-localized polymerase.
Nuphar advena possesses three RpoTgenes
Isolation of Nuphar RpoTcDNAs
Remarkably, NaRpoTp did not exhibit the canonical translation start codon ATG (AUG). Instead, a CTG (CUG) codon was found at position +148, from which translation could be initiated. The following findings are indicative of a translation start from this position: Stop codons in the 5' region exclude further upstream translation initiation sites. The methionine encoded by the most upstream in-frame ATG (nt 466 of NaRpoTp) aligns to amino acid residue 125 of Arabidopsis RpoTp, and the amino terminus derived from this position displayed neither plastid nor mitochondrial targeting properties (see below). On the other hand, the deduced amino acid sequence starting at +148 is enriched in hydroxylated amino acids, but is virtually lacking acidic residues, thus exhibiting features of stroma-targeting plastid transit peptides . Interestingly, a translational start from a CUG codon has been found in the RpoTp gene of tobacco . Thus, we assume that translation of NaRpoTp starts from a non-canonical CUG at position +148.
The predicted NaRpoT proteins comprise 996 (NaRpoTm1), 990 (NaRpoTm2) and 985 (NaRpoTp) amino acids, respectively. NaRpoTm1 and NaRpoTm2 exhibit a remarkably high identity of 96.8%, NaRpoTp has 63.1% and 64.6% identical residues compared with NaRpoTm1 and NaRpoTm2, respectively. The alignment of the RpoT polymerases from N. advena with those from Arabidopsis, Physcomitrella and Selaginella (see Figure 2) demonstrates a high degree of conservation at the amino acid sequence level, most striking in the C-terminal part, including all functionally crucial regions and residues known from the phage T7 RNA polymerase [28, 29].
Targeting of the N. advenaRpoTm1 and RpoTm2 polymerases
NupharRpoTp translation is efficiently initiated at a CUG codon
Examination of NaRpoTp upstream sequences revealed a CTG triplet at nucleotide position +148 (see above). Translation initiation at this CUG codon would give rise to an RpoTp protein of 985 residues, the amino terminus of which was predicted in silico to possess plastid targeting properties. To experimentally test whether translation indeed initiates at this non-canonical codon, the following three Na-RpoTp-GFP constructs were generated (see Figure 3): Na-RpoTpmet*-GFP, with the wild-type CUG (+148) cloned immediately downstream of the 35 S promoter for forced translation; Na-RpoTputr-GFP containing the whole 5' untranslated region of 236 nt and thus preserving the sequence context, known to be crucial for initiation at non-AUG codons in plants ; and Na-RpoTpmut-GFP, in which the CUG was modified to CAC to prevent the recognition of CUG as a startcodon. The Na-RpoTpmet*-GFP construct gave rise to green GFP fluorescence in chloroplasts which overlapped with the red chlorophyll autofluorescence, clearly confirming co-localization of red and green fluorescence in chloroplasts (Figure 4J). An identical fluorescence pattern was observed using construct Na-RpoTputr-GFP (Figure 4K), whereas expression of Na-RpoTp mut -GFP (Figure 4L) completely abolished import of the GFP to the chloroplasts. These data provide convincing evidence that translation of NaRpoTp is solely initiated from the CUG codon at position +148.
Genes encoding phage-type mitochondrial and plastid RNA polymerases have been identified from numerous monocotyledonous and eudicotyledonous angiosperm species (for review, see ). In contrast, knowledge on RpoT polymerases of deep branching land plants is so far limited to the moss Physcomitrella patens [19, 20] and the lycophyte Selaginella moellendorfii , and no information at all is available about phage-type RNA polymerases from the basal angiosperm lineages that precede the monocot-eudicot divergence. Here we show that the waterlily Nuphar advena, a basal angiosperm, encodes three RpoT polymerases. The encoded proteins of 996, 990, and 985 amino acids, respectively, exhibit the characteristic domains that are highly conserved between all RpoT polymerases, including the residues shown to be essential and located within the catalytic pocket of the polymerase (D537, K631, Y639, G640, D812, residue numbers as given for T7 RNA polymerase). The high conservation of amino acid sequences and the identical position of the introns in the RpoT genes of Selaginella, Physcomitrella, Nuphar and monocotyledonous and eudicotyledonous angiosperms (see Figure 2) suggests a common ancestral gene giving rise to all land plant RpoT genes. Phylogenetic analysis (see Figure 5) confirms this hypothesis.
Although Physcomitrella (one mitochondrial and two dual-targeted) and eudictos (one mitochondrial, one plastid and one dual-targeted) possess also three phage-type RNA polymerases, the localization of the three Nuphar RpoT polymerases shows a new pattern. The N-termini of two of the three RpoT genes of N. advena show properties of mitochondrial transit peptides. Using translational fusions of the putative NaRpoT transit peptides with GFP, we demonstrated that these transit peptides confer exclusively mitochondrial import. Mitochondrial import of NaRpoTm1- and NaRpoTm2-GFP was also maintained when the fusion constructs contained the full-length 5'-UTRs of the genes (Figure 4). We included these constructs in our study since the presence of the 5'-UTR may alter the targeting of proteins . Thus, we conclude that N. advena encodes two phage-type mitochondrial RNA polymerases. Phylogenetic analysis (see Figure 5) indicates that the third RpoT gene of Nuphar, NaRpoTp, encodes a plastid phage-type RNA polymerase. In the 5' part of the NaRpoTp cDNA no canonical start codon was identified, with the first ATG triplet occurring only at position 466. However, a potential non-AUG initiation codon (CUG) was revealed at position 148. Translation from this codon would yield an N-terminal leader peptide with genuine plastid targeting properties, as predicted by two prediction algorithms (TargetP and Predotar). Three different GFP fusions were designed to test the translation initiation capacity of this CUG codon. The results proved a plastid import of the derived amino-terminus (Figure 4J), as well as an efficient translation initiation at the CUG within the context of the full-length 5'-UTR (Figure 4K) that could be abolished by modifying the codon to CAC (Figure 4L). Thus, Nuphar RpoTp belongs to the rare cases of non-viral plant genes [35–37] that initiate translation exclusively at a non-AUG codon. Interestingly, this is the second case of non-AUG translation initiation among RpoT genes specifying plastid-localized RNA polymerases: translation of the tobacco RpoTp gene also starts from a CUG codon .
Both mono- and eudicotyledonous plants possess a solely plastid-localized phage-type RNAP (RpoTp) together with a purely mitochondrial-localized RpoT enzyme (RpoTm) and, in the case of eudicots, a third phage-type RNAP with dual localization in both organelles is found. The data presented here suggest that all RpoTp proteins descent from a common duplication event that took place in a common ancestor of all flowering plants. Thus far it is unknown whether ferns or gymnosperms contain nuclear genes encoding plastid-localized phage-type RNAPs as well. Since the duplication event giving rise to the second NEP activity in eudicots is clearly more recent, identification of a purely plastid-localized phage-type RNAP in the basal angiosperm Nuphar advena, orthologous to all other purely plastid-targeted enzymes (RpoTp) of flowering plants, suggests that the acquisition of a nuclear gene-encoded transcriptional activity for plastids, not present in lycopods, took place after the split of lycopods from all other tracheophytes, with or before the rise of flowering plants. Moreover, the lack of a dual-targeted RpoTmp both in Nuphar and in monocots suggests that the RpoTmp enzyme detected in eudicots is an 'invention' due to an RpoTm gene duplication that might have occurred only after the separation of monocots and eudicots. The putative plastid targeting sequences as present in two of the three Physcomitrella RpoT proteins are therefore clearly species- or lineage-specific convergent inventions. Interestingly, multiple mitochondrial RNA polymerasesas as found in Physcomitrella and eudicots are indentified in Nuphar as well. The fixation of duplicated RpoT genes leads to convergent multiplicity of mitochondrial RNAPs in Nuphar, Physcomitrella and eudicots, not found in any other eukaryotic lineage. Recently it was shown that in Arabidopsis RpoTmp null mutants transcription of a specific set of mitochondrial genes is strongly reduced. Moreover, accumulation of respiratory complexes was affected to very different levels, suggesting that the presence of multiple transcriptional activities in mitochondria may allow plants to regulate mitochondrial gene expression in a complex specific manner . Further investigations will be necessary to show if a similar division of labor evolved in case of the two mitochondrial RNA polymerases in Nuphar and address the specific impact of NEP and PEP transcriptional activities for gene expression in Nuphar chloroplasts.
Identification of three RpoT genes in Nuphar advena, specifying two mitochondrial and one plastid-localized polymerases, suggests that multiple phage-type organellar RNAPs already exist among basal angiosperms. From the high similarity of the encoded amino acid sequences, the conservation of intron positions and phylogenetic analysis we conclude that the RpoT genes of Nuphar, like those of Selaginella, Physcomitrella and monocotyledonous and eudicotyledonous angiosperms, trace back to a common ancestral gene giving rise to all land plant RpoT genes. The presence 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 plastid RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that this additional NEP activity (RpoTmp) is an evolutionary novelty of eudicotyledonous plants like Arabidopsis. Our results support the idea that RpoT gene duplications occurred independently of each other several times during the evolution of plants and led to different subcellular localization patterns of of organellar RNA polymerases. These data substantially extend our knowledge about the evolution of the transcriptional machineries in plant organelles.
Plant material and growth conditions
Nuphar advena were purchased from a commercial supplier (Seerosen Shop, Eschede, Germany). The plants were grown in a growth chamber at 23°C with a light/dark regime of 8/16 hr. The intensity of light in all experiments was 210 μmol photons s-1m-2.
DNA and RNA isolation
Leaves of N. advena were ground to fine powder under liquid nitrogen and incubated in three volumes of CTAB buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, 2% β-mercaptoethanol) for 1 hour with agitation at 60°C. The lysate was extracted two times with chloroform-isoamyl alcohol (24:1), and the nucleic acids were precipitated with ethanol. The DNA pellet was washed with 70% ethanol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA). RNA was extracted and purified using the Concert Plant RNA Reagent (Invitrogen, Karlsruhe, Germany) and RNA Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturers' instructions.
Isolation of cDNA and genomic cloning
cDNA cloning, screening of an N. advena BAC library (Nuphar_HindIII BAC; Arizona Genomics Institute, Tucson, AZ) and subcloning were performed according to standard methods . A 1.5 kb cDNA fragment amplified from the 3' part of Selaginella RpoT  was used as a 32P-labelled hybridization probe to screen the Nuphar BAC library, containing 165,888 independent clones on nine individual filters, under non-stringent conditions (58°C). Identified positive clones were purchased from the Arizona Genomics Institute. BAC DNA was isolated using the QIAGEN plasmid midi kit according to the protocol of the manufacturer. Sanger dideoxy sequencing of subclones, or directly of the BAC DNA by primer walking, was performed on an ABI3130xl sequencer (Applied Biosystems, Darmstadt, Germany). From the genomic sequences obtained, primers were designed (for a list of all primers used in the present study, see Additional file 1) for rapid amplification of cDNA ends (RACE). 3'- and 5'- RACE reactions were performed with the RACE primers listed in Additional File 1 using the CapFishing kit (Seegene, Rockville, USA) and Phusion hot start DNA polymerase (Finnzyme, Espoo, Finnland) following the protocols of the manufacturers.
Generation of targeting constructs and transient expression
The amino-terminal sequences were amplified from cDNA of the three N. advena RpoT genes using the primers listed in Additional file 1. Products were ligated into vector pDRIVE (Qiagen) and excised using XbaI and SalI. The fragments were inserted into pOL-GFP  opened with SpeI and SalI, to give the constructs shown in Figure 3. coxIV- and recA-GFP constructs were employed as mitochondrial and plastid control constructs .
All constructs were used to transfect Arabidopsis protoplasts, isolated from 3 - 5 weeks old Arabidopsis leaves grown under long day conditions (23°C, 16/8 hr light/dark), essentially as described . Cell density was adjusted to 2 × 106/ml. 100 μl protoplasts were transfected with 20 μg plasmid DNA in 40% polyethylene glycol 4000, 0.8 M mannitol, 1 mM CaCl2. Transformed protoplasts were examined two days after transfection by confocal laser scanning microscopy with a Leica TCS SP2 using 488 nm excitation and two-channel measurement of emission from 510 to 580 nm (green/GFP) and > 590 nm (red/chlorophyll).
Deduced protein sequences were aligned using ClustalW . Conserved blocks were cut out and merged as described earlier  (see Additional file 2) and subjected to Bayesian, maximum-likelihood and maximum parsimony analysis as implemented in the Geneious program package [42, 43]. The Bayesian inference method employed the Mixed amino acid replacement model with a gamma distribution to represent among-site rate heterogeneity (mixed +γ). MCMC was performed with 1 million generations and four independent chains and two runs. The Markov chain was sampled every 100 generations. Convergence was observed by plots of maximum likelihood (ML) scores and by using the run statistics. The first 20% of all trees generated were discarded; the remaining trees were used to construct a consensus tree and to calculate the posterior branch support values. In addition, maximum likelihood analysis with 1000 and maximum parsimony analysis with 1000 bootstrap replicates were conducted.
We thank Susanne Beick and Björn Richter for their help during the early stage of this study. The excellent technical assistance of C. Stock is gratefully acknowledged. CY was supported by NaFög, Berlin. Part of this work was supported by a grant from the Deutsche Forschungsgemeinschaft (WE 1595/6-2, SFB 429).
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