Identification of RNA Editing Sites in Chloroplast Transcripts from the Maternal and Paternal Progenitors of Tobacco (Nicotiana tabacum): Comparative Analysis Shows the Involvement of Distinct Trans-Factors for ndhB Editing

Tadamasa Sasaki*, Yasushi Yukawa*, Tetsuya Miyamoto{dagger}, Junichi Obokata{dagger} and Masahiro Sugiura*,

* Graduate School of Natural Sciences, Nagoya City University, Mizuho, Nagoya, Japan
{dagger} Center for Gene Research, Nagoya University, Nagoya, Japan

Correspondence: E-mail: sugiura{at}nsc.nagoya-cu.ac.jp.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
RNA editing alters genomic nucleotide sequences at the transcript level. In higher plant chloroplasts, C-to-U conversion is known to occur at around 30 specific sites. The tobacco cultivar Nicotiana tabacum is an amphidiploid derived from ancestors of N. sylvestris (maternal) and N. tomentosiformis (paternal). The chloroplast genome of N. tabacum is believed to originate from an ancestor of N. sylvestris. To study the evolution of RNA editing in higher plant chloroplasts, editing sites in the two likely progenitors have first been identified based on those found in N. tabacum. Altogether 34, 33, and 32 editing sites have been found in the chloroplast transcripts from N. tabacum, N. sylvestris, and N. tomentosiformis, respectively. Thirty-one sites are conserved among the three species, whereas remarkable differences are observed in the editing of ndhB and ndhD transcripts. Sites 7 and 8 in ndhB mRNAs are separated only by five nt, and both are edited in N. tabacum and N. sylvestris. However, site 8 is not edited in N. tomentosiformis, indicating that distinct trans-factors are involved in the two editing events. The first site in ndhD mRNAs is edited to produce an AUG start codon in N. sylvestris as well as in N. tabacum but not in N. tomentosiformis, suggesting that a distinct mechanism operates for the translational initiation of N. tomentosiformis ndhD mRNAs. Four to six sites are edited partially in green leaves. Some of these sites may represent evolutionary intermediates in the process of losing editing events.

Key Words: chloroplast • RNA editing • trans-factor • transcript • tobacco


    Introduction
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
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 Literature Cited
 
The chloroplast is known to possess its own genome and gene expression system (Sugiura 1992). Many chloroplast genes in land plants are transcribed as polycistronic RNAs, which are then processed into mature RNA species via complex pathways including RNA editing and splicing (Sugiura, Hirose, and Sugita 1998; Rochaix 2001). RNA editing in chloroplasts was first reported in the maize rpl2 mRNA (Hoch et al. 1991), and a systematic search of chloroplast transcripts identified 27 editing sites in maize (Maier et al. 1995; Bock, Hermann, and Fuchs 1997), 26 editing sites in black pine (Wakasugi et al. 1996), 31 editing sites in tobacco (Hirose et al. 1999), and 21 editing sites in rice (Corneille, Lutz, and Maliga 2000). Editing in chloroplasts occurs generally in protein-coding regions and restored evolutionary conserved amino acid sequences (Maier et al. 1996). However, editing at the third position of a codon (silent editing) and editing in an untranslated region has also been reported (Hirose et al. 1996; Kudla and Bock 1999). In addition, extensive RNA editing, both C-to-U and U-to-C changes have been reported in the chloroplast of hornwort Anthoceros formosae (Yoshinaga et al. 1996). RNA editing has been found in chloroplast transcripts from all major lineages of land plants; however, neither frequency of editing nor the pattern of editing a specific transcript correlate with the phylogenic tree of the plant kingdom (Freyer, Kiefer-Meyer, and Kössel 1997).

A key question in chloroplast editing is how specific C residues are recognized precisely from all other C residues in transcripts. Using transgenic approaches in tobacco chloroplasts, cis-acting elements have been analyzed for psbL mRNAs (Chaudhuri, Carrer, and Maliga 1995; Chaudhuri and Maliga 1996), for ndhB mRNAs (sites 4 and 5) (Bock, Hermann, and Kössel. 1996; Bock, Hermann, and Fuchs 1997; Hermann and Bock 1999), and for ndhF and rpoB (site 2) mRNAs (Reed, Lyi, and Hanson 2001). These studies commonly showed that cis-acting elements reside in upstream regions of the editing sites. Furthermore, chloroplast transplastomic experiments suggested the involvement of trans-acting factors in editing (Chaudhuri, Carrer, and Maliga 1995; Chaudhuri and Maliga 1996; Bock and Koop 1997; Reed and Hanson 1997; Reed, Lyi, and Hanson 2001; Schmitz-Linneweber et al. 2001). These in vivo analyses show that at least some trans factors appear to be site specific and of extraplastidic origin. Recently, an in vitro RNA editing system from tobacco chloroplasts was developed in our laboratory to dissect biochemical processes of editing reactions in chloroplasts (Hirose and Sugiura 2001). Using this system, a tobacco chloroplast protein of 25 kd was found to bind specifically to the cis-acting element of psbL mRNA. This result provided the evidence that the protein, but not RNA, is the trans-acting factor that is likely to recognize the editing site of psbL mRNAs. An improved method was then reported for preparing chloroplast extracts supporting accurate RNA editing reactions in vitro not only from tobacco but also from pea (Miyamoto, Obokata, and Sugiura 2002). Using this improved system, we defined cis elements of psbE and petB mRNAs and detected trans factors that specifically bind to these elements, 56-kd and 70-kd proteins for psbE and petB mRNAs, respectively.

In the case of tobacco chloroplasts, the genome sequence has been completely determined (Shinozaki et al. 1986), the gene organization has been updated (Wakasugi et al. 1998; Wakasugi, Tsudzuki, and Sugiura 2001), and a systematic search for editing sites in the transcripts has been made (Hirose et al. 1999). In addition, both chloroplast transformation techniques (in vivo) (Svab and Maliga 1993) and chloroplast RNA editing system (in vitro) (Hirose and Sugiura 2001) are available only for tobacco. Therefore, tobacco is the organism of choice for analyzing detailed mechanisms of RNA editing in chloroplasts. The tobacco cultivar Nicotiana tabacum is a natural amphidiploid derived from two progenitors, and ancestors of N. sylvestris and N. tomentosiformis were the likely progenitors of N. tabacum (Smith 1974; Kenton et al. 1993). The chloroplast genome of N. tabacum is believed to have originated from N. sylvestris (Olmstead and Palmer 1991). Hence, these Nicotiana species offer a significant advantage for evolutional studies of RNA editing in higher plant chloroplasts. Recently, Schmitz-Linneweber et al. (2001) reported an interesting observation that N. tabacum lost an editing site but still possesses its trans factor, which probably originated from a progenitor of N. tomentosiformis.

Here we report the editing pattern of both N. sylvestris and N. tomentosiformis chloroplasts. Comparative analysis with editing sites in N. tabacum shows the opposite case as above, namely the presence of a C residue (to be edited in N. tabacum and N. sylvestris) but no corresponding editing activity in N. tomentosiformis. This analysis also indicates the involvement of distinct trans factors for two adjacent sites.


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
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 Literature Cited
 
Nicotiana tabacum (var. Bright Yellow 4), N. sylvestris, and N. tomentosiformis leaves were harvested from 6-week-old plants grown in a growth chamber at 28°C under 16 h light/8 h dark conditions. Total cellular DNA and RNA were isolated from green leaves essentially as described by Wakasugi et al. (1994), with minor modifications. cDNA synthesis was carried out according to the instruction manual of the High Fidelity RNA PCR kit (TaKaRa) using 1 µg of total cellular RNA as template. Sequences encompassing editing sites were amplified by PCR from the cDNA and total cellular DNA using gene-specific primers listed in table 1, purified with GFXTM PCR DNA and Gel Band Purification kit (Amersham Pharmacia), and sequenced using the BigDye Terminator Cycle Sequencing FS Ready Reaction kit (ABI). Computer analysis of editing sites was carried out using GENETYX-MAC version 9.0 (Software Development Co.) and Sequencher version 3.0 (Gene Codes Co.).


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Table 1 PCR Primers Used in This Work.

 

    Results and Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
RNA Editing Sites in Chloroplast Transcripts from Nicotiana Species
The N. tabacum chloroplast genome has so far been found to include 80 different protein-coding genes and 35 different genes encoding stable RNA species (Wakasugi et al. 2001) and 31 editing sites (all C-to-U conversion) which were observed from 16 gene transcripts (Hirose et al. 1999). Based on information from these sites, direct sequencing was performed for PCR-amplified cDNA fragments (145 to 1,869 bp) derived from N. sylvestris and N. tomentosiformis transcripts. During the course of this study, three additional C-to-U editing sites were found in N. tabacum, one from rps2 mRNAs (site 1) and two in ndhD mRNAs (sites 3 and 4). Therefore, a total of 34 sites were surveyed. Table 2 compiles the editing sites identified in the three Nicotiana species. All the 34 sites are conserved at the DNA level (all C residues) among the three species, whereas no editing was observed at the transcript level for one site (atpA site 2) in N. sylvestris and three sites (atpA site 2, ndhB site 8, and ndhD site 1) in N. tomentosiformis. An additional editing site unique to ndhA mRNAs from N. tomentosiformis (already a T at this site in N. tabacum and N. sylvestris) was reported by Schmitz-Linneweber et al. (2001). Therefore, total numbers of editing sites so far identified are 33 in N. sylvestris and 32 in N. tomentosiformis.


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Table 2 RNA Editing Sites in Nicotiana Chloroplast Transcripts.

 
Editing of ndhB Transcripts
The ndhB gene coding for NADH dehydrogenase subunit II is located within the large inverted repeat of the N. tabacum chloroplast genome and produces mature spliced mRNAs of ca. 1.5 kb (Matsubayashi et al. 1987). Nine editing sites have been identified in the transcript from the N. tabacum ndhB gene (Freyer, Kiefer-Meyer, and Kössel 1997; Hirose et al. 1999), the highest frequency in a single mRNA species. As five additional protein-coding genes are positioned within the repeat and no editing has been reported in their transcripts, high editing frequency of ndhB transcripts is not due to its gene location.

As shown in figure 1A, ndhB sites 7 and 8 in N. tabacum are separated only by 5 nt (one alanine codon, GCU) and both editing events cause an amino acid substitution from serine (UCA) to leucine (UUA). This is also the case for N. sylvestris, whereas editing was not observed in N. tomentosiformis for the C residue corresponding to site 8 of N. tabacum (fig. 1B). cDNA sequencing was repeated three times with different RNA preparations, and the same results were obtained. Therefore, we concluded that N. tomentosiformis lacks editing activity for site 8 despite conserved sequences around this position among the three Nicotiana species. On the other hand, another pair of ndhB sites 5 and 6, separated only by 8 nt, are edited in N. tomentosiformis as in N. tabacum and N. sylvestris (fig. 1B). These results indicate that site-recognition factors for sites 7 and 8 are different, because editing should occur in both sites if a single factor recognizes both C residues to be edited. This is consistent with the observation on transplastomic tobacco lines that the ndhB sites 7 and 8 are edited independently (Bock, Hermann, and Fuchs 1997).



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FIG. 1. RNA editing of ndhB transcripts. (A) Schematic representation of the N. tabacum ndhB gene. Numbers at the top indicate editing sites. Nucleotide positions are from the A (+1) of its start codon. Partial sequences around sites 5 and 6 and sites 7 and 8 are shown below. Arrows indicate C-to-U conversion. (B) Sequence analysis of three cDNAs around sites 7 and 8 (indicated by arrows)

 
Editing of ndhD (Site 1) Transcripts
The ndhD gene encodes NADH dehydrogenase subunit IV and is located in the middle of the small single-copy region of the N. tabacum chloroplast genome (Shinozaki et al. 1986). This gene is cotranscribed with the upstream psaC, and the resultant dicistronic pre-mRNA of approximately 2.5 kb is processed into monocistronic forms of approximately 2 kb (ndhD) and approximately 0.5 kb (psaC) in N. tabacum chloroplasts (Matsubayashi et al. 1987; Hayashida et al. 1987). The initiation codon of the N. tabacum ndhD gene was believed to be the ATG located 95 bp downstream from the psaC coding region, and a Shine-Dalgarno (SD)-like sequence (GAG) 12 bp upstream from the ATG was assigned as a potential ribosome binding site (fig. 2A; Matsubayashi et al. 1987). However, C-to-U editing has been shown to occur at ACG 25 nt downstream from the AUG in the N. tabacum ndhD transcript, which leads to the creation of an in-frame AUG codon (Neckermann et al. 1994). Our chloroplast in vitro translation assay has indicated that only the edited AUG codon acts as the actual initiation codon of N. tabacum ndhD mRNAs and that the upstream AUG has no function as an initiation codon (Hirose and Sugiura 1997). Recently, the leek ndhD transcript was reported to require editing to restore its start codon, which may be used as a marker for the processing of psaC and ndhD transcripts (Del Campo et al. 2002).



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FIG. 2. RNA editing of ndhD transcripts. (A) Schematic representation of the N. tabacum psaC-ndhD operon. Numbers at the top indicate editing sites. Nucleotide positions are from the A (+1) of its start codon. A partial sequence including the spacer is shown below. The sequence is identical in N. tabacum and N. sylvestris but differ in two positions -50 (C to A) and -49 (A to C) in N. tomentosiformis. Coding regions are boxed. Underlines show potential start codons. Arrow indicates C-to-U conversion. (B) Sequence analysis of two cDNA around site 1 (indicated by arrows)

 
Our cDNA analysis showed that the ACG codon is also edited to produce AUG in N. sylvestris. However, editing of the ACG at the same position was not observed in N. tomentosiformis (fig. 2B). The experiment was repeated two more times with different RNA preparations and the same negative results were obtained. Therefore, the mechanism of translational initiation for ndhD mRNAs is likely to be different between N. tomentosiformis and N. tobacum. N. tomentosiformis chloroplasts may possess a factor that can allow initiation from the upstream AUG codon, or its translation may start from the in-frame GUG codon 6 nt downstream from the AUG triplet (see fig. 2A). However, it cannot be ruled out that a functional ndhD gene is present in the nucleus of N. tomentosiformis.

Silent Editing in atpA Transcripts
The atpA gene encodes the {alpha}-subunit of ATP synthase complex and is located at the last gene in the rps2-atpIHFA operon (Wakasugi et al. 1998). Transcription of this operon in N. tabacum starts from at least four sites, and resulting polycistronic mRNAs are processed from at least four sites to produce a dicistromic atpF-atpA mRNA and other mRNAs (Miyagi et al. 1998). As shown in figure 3A, two editing sites were found in two successive codons of N. tabacum atpA transcripts (Hirose et al. 1996). The first C-to-U conversion caused proline (CCC) to leucine (CUC) substitution, whereas the second editing took place partially at the third position of the serine codon (UCC to UCU), leading to no amino acid change (silent editing). In the case of N. sylvestris and N. tomentosiformis atpA mRNAs, editing was detected in the first codon but not in the second position (fig. 3B), as was found in pea atpA mRNAs (Hirose et al. 1996). Therefore, silent editing at the serine codon seems to be unique to N. tabacum.



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FIG. 3. RNA editing of atpA transcripts. (A) Schematic representation of the N. tabacum rps2-atpIHFA operon. Numbers at the top indicate editing sites. A partial sequence around sites 1 and 2 is shown below. Arrows indicate C-to-U conversion. (B) Sequence analysis of two cDNAs around sites 1 and 2 (indicated by arrows)

 
Partial Editing in Green Leaves
Most transcripts are essentially completely edited, whereas a limited number of editing sites are edited partially, suggesting that certain editing events play a regulatory role in gene expression (Bock et al. 1993; Hirose et al. 1996, 1999; Hirose and Sugiura 1997; Ruf and Kössel 1997; Nakajima and Mulligan 2001; Karcher and Bock 2002). Recently, a comprehensive analysis of the editing efficiency of each of the 27 known sites in maize chloroplasts was performed (Peeters and Hanson 2002). Editing efficiencies of some sites were found to be affected by the developmental stage, quite low in roots and calli, whereas editing of all 27 sites in young green leaves is close to 100% efficiency.

We used transcripts from young green leaves to identify editing sites in the Nicotiana species. Most identified sites are fully edited, whereas a limited number of identified sites are edited partially (table 2). Partial editing was confirmed by three independent cDNA sequencing from different RNA preparations. The extent of editing is dependent on the site. In N. tabacum, partial editing was reported in atpA site 2 (Hirose et al. 1996), ndhD site 1 (Hirose and Sugiura 1997) and rpoA (Hirose et al. 1999). An additional site, ndhD site 3, was found from our own analysis to be partially edited (fig. 4). Altogether four sites are partially edited in N. tabacum green leaves.



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FIG. 4. Partial RNA editing of ndhB, ndhD and rpoC1 transcripts. Sequence analysis of cDNAs around editing sites where editing is partial. Arrows indicate editing sites

 
In N. sylvestris and N. tomentosiformis green leaves, six sites were found to be edited partially, of which four sites are common (ndhB sites 3 and 4, ndhD site 3, and rpoC1) between them (fig. 4). Editing at ndhD site 1 was partial in N. sylvestris (see fig. 4), similar to that in N. tabacum (Hirose and Sugiura, 1997). As this editing creates AUG from ACG, approximately half of the ndhD mRNA seems to be nonfunctional in N. sylvestris green leaves, as in the case of N. tabacum. The ndhD site 4 from N. tomentosiformis was edited slightly, but the site was edited fully in the other two species (fig. 4; Hirose et al. 1999).

Editing of ndhB sites 3 and 4 and rpoC1 occurs fully in N. tabacum (Hirose et al. 1999) but not in the other species. This phenomenon could be explained by doubling nuclear genes encoding editing factors. Recently, it was reported that editing of the two ndhB sites in N. tabacum is temperature sensitive (Karcher and Bock 2002). Therefore, interaction of cis elements with editing machineries for these editing sites may be more fragile when compared with other sites. The corresponding sites in maize plastids were poorly edited in roots and calli (Peeters and Hanson 2002), suggesting low expression of the trans factors in nonphotosynthetic cells. However, the opposite case was observed for rpoA editing; partial in the tetraploid but full in the two diploid species. This may be due to interference in N. tabacum between the expression of nuclear genes encoding the editing factors or between these factors derived from the two progenitors. Partial editing implies the presence of two or more different mRNA species from single genes, which potentially leads to the microheterogeneity of relevant protein products. If this is not the case, mRNA surveillance mechanisms should operate in chloroplasts. In the case of an editing event creating AUG start codons, unedited mRNAs are most likely nonfunctional.

Conclusion
Recent findings show that an editing activity can be present despite the absence of the target site; N. tabacum is capable of editing the exogenous ndhA site 2 even though its plastid genome lacks this site (Schmitz-Linneweber et al. 2001). The nucleus of N. tomentosiformis is suggested to be the donor of the corresponding trans factor. Here we present evidence to the contrary; N. tabacum and N. sylvestris have ndhB site 8 and ndhD site 1 and their cognate editing activities, whereas no editing activity for these sites in N. tomentosiformis was detected even though these positions hold C residues in their genome. Therefore, these editing factors are thought to originate from N. sylvestris. As for the ndhB site 8, the ndhB protein may be functional with either serine or leucine at this position. If this is the case, editing of this site is dispensable and N. tomentosiformis lost its editing activity. This suggests that conserved amino acid residues are not always essential for protein function. Comparison of editing patterns among N. tabacum (amphidiploid) and its progeny representatives, N. sylvestris and N. tomentosiformis, will provide clues for better understanding of the evolution of editing events in plastids.


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 Abstract
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The sequences reported here have been deposited in the DNA Databank of Japan (DDBJ, accession numbers AB098210 to AB098251).


    Acknowledgements
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 Abstract
 Introduction
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 Supplementary Material
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We thank T. Tsudzuki, T. Wakasugi, T. Nakamura, and K. Hasegawa for continuous discussions and suggestions. This work was supported in part by a Grant-in-Aid for Scientific Research in Priority Areas C (No. 13201001) from the Ministry of Education, Science, Sports and Culture.


    Footnotes
 
Kenneth Wolfe, Associate Editor Back


    Literature Cited
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 Introduction
 Materials and Methods
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 Supplementary Material
 Acknowledgements
 Literature Cited
 

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Accepted for publication January 14, 2003.