Mitochondrial Gene Transfer in Pieces: Fission of the Ribosomal Protein Gene rpl2 and Partial or Complete Gene Transfer to the Nucleus

Keith L. Adams, Han Chuan Ong and Jeffrey D. Palmer

Department of Biology, Indiana University, Bloomington


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Mitochondrial genes are usually conserved in size in angiosperms. A notable exception is the rpl2 gene, which is considerably shorter in the eudicot Arabidopsis than in the monocot rice. Here, we show that a severely truncated mitochondrial rpl2 gene (termed 5' rpl2) was created by the formation of a premature stop codon early in eudicot evolution. This 5' rpl2 gene was subsequently lost many times from the mitochondrial DNAs of 179 core eudicots surveyed by Southern hybridization. The sequence corresponding to the 3' end of rice rpl2 (termed 3' rpl2) has been lost much more pervasively among the mitochondrial DNAs of core eudicots than has 5' rpl2. Furthermore, where still present in these mitochondrial genomes, 3' rpl2 always appears to be a pseudogene, and there is no evidence that 3' rpl2 was ever a functional mitochondrial gene. An intact and expressed 3' rpl2 gene was discovered in the nucleus of five diverse eudicots (tomato, cotton, Arabidopsis, soybean, and Medicago). In the first three of these species, 5' rpl2 is still present in the mitochondrion, unlike the two legumes, where both parts of rpl2 are present in the nucleus as separate genes. The full-length rpl2 gene has been transferred intact to the nucleus in maize. We propose that the 3' end of rpl2 was functionally transferred to the nucleus early in eudicot evolution, and that this event then permitted the nonsense mutation that gave rise to the mitochondrial 5' rpl2 gene. Once 5' rpl2 was established as a stand-alone mitochondrial gene, it was then lost, and was probably transferred to the nucleus many times. This complex history of gene fission and gene transfer has created four distinct types of rpl2 structures or compartmentalizations in angiosperms: (1) intact rpl2 gene in the mitochondrion, (2) intact gene in the nucleus, (3) split gene, 5' in the mitochondrion and 3' in the nucleus, and (4) split gene, both parts in the nucleus.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Mitochondrial genomes are derived from the genome of a bacterial endosymbiont, the majority of whose genes were lost or transferred to the nucleus early in mitochondrial evolution (reviewed in Gray 1992Citation ; Gray, Burger, and Lang 1999Citation ). Mitochondrial gene content varies considerably (from 3 to ~60 protein genes) across the broad sweep of eukaryotes (Gray et al. 1998Citation ; Lang, Gray, and Burger 1999Citation ); this largely appears to reflect different degrees of gene transfer to the nucleus. Although no cases of recent mitochondrial gene transfer and functional activation have been reported in animals, numerous mitochondrial pseudogenes have been discovered in the nucleus of a variety of animals (reviewed in Bensasson et al. 2001)Citation . In contrast to animals, a number of cases of functional mitochondrial gene transfer have been characterized in plants (e.g., Adams et al. 1999Citation ; Figueroa et al. 1999Citation ; Kubo et al. 1999, 2000aCitation ; Palmer et al. 2000Citation and references therein) and green algae (Pérez-Martínez et al. 2000, 2001Citation ), including many separate transfers of the same gene in angiosperms (Adams et al. 2000, 2001aCitation , 2001b). Sporadically repeated gene transfer to the nucleus has led to a highly variable distribution of ribosomal protein and succinate dehydrogenase genes among the mitochondrial genomes of angiosperms.

Although mitochondrial gene content is highly variable in angiosperms, the sizes of these genes are generally well conserved. Two exceptions to this pattern are the cytochrome c biogenesis gene ccb577 (equivalent to the N-terminal portion of the bacterial ccl1 gene) and the ribosomal protein gene rpl2. Ccb577 has been characterized from the mitochondrion of six diverse angiosperms. In four cases (Oenothera, carrot, wheat, and beet), the gene is present as a single open reading frame (ORF) (Gonzalez, Bonnard, and Grienenberger 1993Citation ; Schuster et al. 1993Citation ; Kubo et al. 2000bCitation ). However, in the crucifers Brassica and Arabidopsis, ccb577 is split into two widely separated and independently transcribed ORFs (Handa, Bonnard, and Grienenberger 1996Citation ; Unseld et al. 1997Citation ). Thus, the ccb577 gene was split relatively recently during eudicot evolution, as discussed by Handa, Bonnard, and Grienenberger (1996)Citation .

The locus for the ribosomal protein gene rpl2 has been sequenced from the mitochondrial DNA of five flowering plants. In both Arabidopsis (Sunkel, Brennicke, and Knoop 1994Citation ; Unseld et al. 1997Citation ) and Oenothera (EMBL accession number X80170), rpl2 is missing about 550 bp at the 3' end of the gene that are present in rpl2 from rice (Kubo et al. 1996Citation ), the liverwort Marchantia (Oda et al. 1992Citation ), and the protist Reclinomonas (Lang et al. 1997Citation ). The missing region is not present elsewhere in the completely sequenced Arabidopsis mitochondrial genome (Unseld et al. 1997Citation ), but it is present in the nucleus of Arabidopsis (EMBL accession number X82556).

Wheat mitochondrial DNA contains only a small fragment of rpl2, from the 3' end of the gene, and the rpl2 fragment is cotranscribed with another gene (Subramanian, Fallahi, and Bonen 2001Citation ). rpl2 has been transferred to the nucleus in wheat, where two copies are expressed (Bonen, Carrillo, and Subramanian 1998Citation ). The first exon of rpl2 has been sequenced from the mitochondrion of potato (Loessel et al. 1999Citation ), although the sequenced region does not extend to the 3' end of the gene.

In this study we show that the 5' rpl2 gene in mitochondrial DNA was created by the formation of a stop codon in the common ancestor of core eudicots, and that this event was probably allowed by the slightly earlier transfer of the 3' end of rpl2 to the nucleus and its establishment there as a functional gene. The two resulting functional rpl2 genes are present in different cellular compartments in some core eudicots, and in the same compartment (the nucleus) in others.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Nucleic acid isolations, Southern blots, and hybridizations were performed as described in Adams et al. (2001b)Citation , using filters with BamHI-digested DNAs that were used for surveys of sdh3 and sdh4 (Adams et al. 2001bCitation ). See http://www.bio.indiana.edu/palmerlab for species names and voucher information. Templates for rpl2 probes were generated by PCR amplification using the following primers: 5' rpl2 (5'-ATGCCTCCTTTGTCTTCGTC-3' and 5'-AGTTACCTTGATCCGTCTTC-3') and 3' rpl2 (5'-TTGAAAAACTCATAGATTCCCG-3' and 5'-GTGGGGAAACGCACAATTTAG-3') for Arabidopsis and rice DNAs, respectively. PCR conditions were as described in Adams et al. (2001b)Citation .

The mitochondrial 3' rpl2 sequence was PCR-amplified from several core eudicot DNAs using the following primers: forward (5'-GAAGCRGGYAADATGGTGAT-3') and reverse (5'-GTGGGGAAACGCACAATTTAG-3). The mitochondrial 5' rpl2 gene was PCR-amplified from several core eudicot genomic DNAs or cDNAs using the primer F1 (5'-GAGCRCTTAGACAWTTCACT-3') and one of the following reverse primers: R1 (5'-ACAGGTGCACGATCKACTTTG-3'), R2 (5'-GRCGTGGCCAAGAAGCAGC-3'), or R4 (5'-CYGCCCAGAATGCYCATMCAT-3'). Most of exon 2 was PCR-amplified from three basal eudicot DNAs using the primers F1 (5'-GAGCRCTTAGACAWTTCACT-3') and Rev (5'-GTGGGGAAACGCAGAATTTAG-3'). All PCR products were sequenced directly on both strands using an ABI 3700 DNA sequencer. Mitochondrial rpl2 sequences determined in this study have been assigned GenBank accession numbers AF387174AF387188.

EST clones of nuclear 3' rpl2 from Arabidopsis (AW004239) and soybean (BE473673) were purchased from Incyte Genomics and sequenced using M13 forward and reverse primers; maize rpl2 EST AW066220 was purchased from the ZmDB maize EST–sequencing project and completely sequenced using M13 primers and the following internal primers: (5'-AAGCCAAAGCGGATGAAGAAG-3' and 5'-CTTTCTTAGCTTGCGTGTACC-3'). The genomic sequences of nuclear 3' rpl2 from cotton, tomato, and soybean were obtained by PCR amplification and sequencing using the following primers: cotton (5'-GAGGGCGCACAGCGTCTTTGA-3' and 5'-TAARWCTCGAMTTACGGTTTAG-3'), tomato (5'-CTCGAATGGCTTCATCTTTCC-3' and 5'-GGATACTTAATTGTCAATGTTATG-3'), and soybean (5'-GAAATGGCTGTGTCACTGTGG-3' and 5'-CCAACTGCCCTCGGGTGTTA-3'). The genomic sequence of the nuclear 5' rpl2 gene from soybean was PCR-amplified using the primers (5'-GAAGAAAAGAGTGTAAGAGTAG-3') and (5'-TGCTTTGCCACGAAATCGCTG-3'). Nuclear rpl2 sequences determined in this study have been assigned GenBank accession numbers AY044158AY044160.

Accession numbers for additional nuclear EST and Arabidopsis genome sequences utilized in this study are as follows: soybean 5' rpl2 (BG406182, AW706585, BF071707), Medicago 5' rpl2 (BE941643 and BF646066), Lotus 5' rpl2 (AV422504 and AV424117), Arabidopsis 3' rpl2 genomic sequence (AC004005), cotton 3' rpl2 (BE054892 and BF269312), tomato 3' rpl2 (AW626051 and BE460252), and Medicago 3' rpl2 (BF646435 and AL373876).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Many Absences of the 5' and 3' Regions of rpl2 from Angiosperm Mitochondrial DNAs
To comprehensively survey the presence or absence of the 5' and 3' regions of rpl2 (see fig. 1 ) in angiosperm mitochondria, Southern blot hybridizations of total DNAs from 280 diverse angiosperms were performed. Such a wide hybridization survey was feasible because of the very low nucleotide substitution rate of angiosperm mitochondrial genes (Wolfe, Li, and Sharp 1987Citation ; Laroche et al. 1997Citation ). Two probes were used: a 399-bp region of 5' rpl2 exon 1 and the last 269 bp of 3' rpl2 (fig. 1 ). Mitochondrial gene loss was inferred if there was no detectable (or in two cases, severely diminished) hybridization on an overexposed autoradiograph against two controls: good hybridization to the DNA in question using other probes (e.g., nad9; fig. 2 ) and good hybridization to other DNAs with the rpl2 probe. This inference assumes that mitochondrial genes are in high copy number and conserved in sequence relative to the transferred nuclear genes of single or low copy number and divergent sequence. The 5' and 3' probes failed to hybridize to 88 and 130, respectively, of the 280 DNAs examined, a sampling of which is shown in figure 2 . In addition, both exons of rpl2 (see fig. 1 ) were previously inferred to be missing from pea mitochondrial DNA (not included on our blots) based on Southern hybridization with a probe from rice rpl2 (Kubo et al. 1996Citation ), and rpl2 was reported to be missing from soybean mitochondrial DNA (Bonen, Carrillo, and Subramanian 1998Citation ).



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Fig. 1.—Structure of mitochondrial rpl2, approximate sizes of regions of rpl2 in mitochondrial DNA, gene nomenclature used in this study, and locations of the probes used for Southern hybridizations

 


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Fig. 2.—Southern blot hybridizations of total DNAs from 38 angiosperms (of the 280 examined in total) with the mitochondrial gene probes indicated. Ovals indicate absence of hybridization to the 5' rpl2 probe, and rectangles indicate absence of hybridization to the 3' rpl2 probe. The hybridization of the 3' rpl2 probe to Cucumis and Datisca DNAs was judged to be severely diminished relative to controls and was therefore scored as an absence

 
The presence and absence data were mapped separately for each probe on to a phylogenetic tree of the surveyed species (fig. 3 ). A total of 41 separate losses of 5' rpl2 were inferred. Most losses are found sporadically distributed among the eudicots and monocots, but there are few losses in basal angiosperms. Some losses appear to be very recent, for example, those in the asterids Phlox and Galium (fig. 3 , bottom right), whereas other losses encompass larger phylogenetic groups, such as the losses in the Asterales and Apiales (fig. 3 , middle right). Overall, the general pattern and phylogenetic timing of 5' rpl2 losses resemble those of several other mitochondrial genes (Adams et al. 2000, 2001a, 2001bCitation ).



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Fig. 3.—Many losses of 5' and 3' regions of mitochondrial rpl2 among the 280 angiosperms surveyed and summary of mitochondrial rpl2-sequencing data. Columns of ovals and rectangles indicate absence of hybridization on the Southern blots. Bold lettering and larger type indicate species from which mitochondrial or nuclear rpl2 sequences (or both) are available (see text). Pseudo symbols ({Psi}) indicate sequenced pseudogenes; these are also highlighted by small arrows. The term, entire ORF, indicates that an entire rpl2 ORF (see fig. 1) is found in the mitochondrion. Plus signs indicate 5' rpl2 sequences in mitochondrial DNA that were obtained in this study (Gossypium, Ulmus, and Lonicera) or from GenBank (Arabidopsis and Oenothera). The phylogenetic tree is from Adams et al. (2001aCitation , 2001b) and is based largely on the strict consensus trees from Soltis, Soltis, and Chase (1999)Citation and Soltis et al. (2000)Citation , although other studies were also referred to (see http://www.bio.indiana.edu/palmerlab). Common names used in the text and other figures include cotton (Gossypium), tomato (Lycopersicon), soybean (Glycine), rice (Oryza), and maize (Zea)

 
Among certain groups of angiosperms, the hybridization pattern of the 3' rpl2 probe is markedly different from that of the 5' probe. In basal angiosperms, monocots, and basal eudicots, the two hybridization patterns are fairly similar, but among most core eudicots, i.e., asterids and caryophyllids (fig. 3 , right), and rosids and Saxifragales (fig. 3 , middle), many more taxa failed to hybridize with the 3' probe than with the 5' probe. A very large number of losses of the 3' region would be inferred from the hybridization data if each separate clade with no hybridization were scored as a separate loss, as most core eudicot DNAs with 3' absences are closely interspersed phylogenetically with DNAs to which the 3' probe hybridized. This raises the possibility that the 3' rpl2 absences within these groups are the result of one or a few deeper losses of function, with only mitochondrial pseudogenes surviving in some or many of the relatively few, sporadically distributed core eudicots showing 3' hybridization.

Truncation of Mitochondrial rpl2 at the Base of Core Eudicots
To determine how and when the 5' rpl2 ORF was formed, a region of exon 2 spanning the sites of the 5' ORF termination codons in Arabidopsis and Oenothera (the stop codons are not in the same position in these two species) and including most of 3' rpl2 (see fig. 1 ) was PCR-amplified and sequenced from 12 diverse eudicots. In Dicentra, Platanus, and Trochodendron (three members of the basal eudicot grade), the entire sequenced region was an intact ORF (i.e., the termination codons present in Oenothera and Arabidopsis were absent—see fig. 4 ). Thus, the structure of rpl2 in the mitochondrion of Dicentra, Platanus, and Trochodendron appears to be one entire ORF (probably interrupted by an intron), as in rice (see fig. 1 ). The same region was also isolated and sequenced from nine core eudicots belonging to groups in which there was hybridization of the DNAs to the 3' rpl2 probe. The sequences from all nine taxa—the asterids Lonicera, Viburnum, Diospyros, Hydrangea, Chamaedaphne, and Cornus (fig. 3 , right), the rosids Quercus and Abutilon (fig. 3 , middle), and Corylopsis from the Saxifragales (fig. 3 , middle)—contain a stop codon (TAA) at the same position as the 5' ORF termination codon in Oenothera (fig. 4 ). Thus, the gene fission event that created the 5' rpl2 ORF/gene seems to have occurred via a C-to-T terminator mutation in the common ancestor of core eudicots, after the divergence of the Trochodendraceae (fig. 3 , large gray arrow). An unlikely alternative is that a rare U-to-C RNA editing event might restore the CAA codon in the mRNA. Very few U-to-C edits (only four out of the hundreds of known edits) have been documented in flowering plants (reviewed in Maier et al. 1996Citation ), and there are no U-to-C edits among the 441 RNA editing sites in the mitochondrial mRNAs of Arabidopsis (Giegé and Brennicke 1999Citation ). Even if the TAA codon were corrected by RNA editing, the rpl2 gene would be truncated in eight of the nine species examined by premature stop codons, and frameshifts would disrupt the reading frame in several species, as discussed later.



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Fig. 4.—Alignment of mitochondrial rpl2 nucleotide sequences flanking the point of gene fission. Sequences are aligned to rice; dots indicate nucleotides identical to rice. Dashes indicate gaps inserted to improve alignment. Bold lettering indicates the codon at which a C-to-T change has created a stop codon in all species except Arabidopsis; the Arabidopsis rpl2 stop codon is located downstream of this (as a result of a 4-bp deletion immediately upstream of the C/T nucleotide) and is also bolded

 
There is no evidence for a methionine codon that might serve as a start codon for a 3' rpl2 ORF in any of the nine core eudicot 3' rpl2 sequences. The first ATG codon in 3' rpl2 is about 230 bp downstream of the end of the 5' ORF; if used as a start codon, the 3' ORF would be truncated by about 76 amino acids (aa), about 45 of which are in a highly conserved region of rpl2 relative to other eukaryotes. A start codon could not be generated by the C-to-U RNA editing that commonly occurs in plant mitochondria (reviewed in Maier et al. 1996Citation ) because there are no ACG codons in the correct reading frame in the first two-thirds of 3' rpl2. Furthermore, most of the 3' rpl2 sequences have one or more in-frame stop codons that would disrupt a reading frame. In some cases the premature stop codons are created by terminator point mutations (Quercus and Viburnum), whereas in other cases deletions or insertions (indels) would disrupt a reading frame (small indels in Chamaedaphne, Diospyros, Corylopsis, Cornus, and Lonicera and a deletion of over 100 bp in Abutilon 3' rpl2). Only Hydrangea 3' rpl2 contains no premature stop codons or indels that would disrupt a reading frame. Taken together, these observations suggest that 3' rpl2 probably never existed as a functional ORF in plant mitochondria, contrary to our initial expectations.

In contrast to the 3' rpl2 pseudogenes in the mitochondrial DNA of several core eudicots, exon 1 of 5' rpl2 (most of the 5' end; fig. 1 ) is intact in the mitochondrion of Arabidopsis, Oenothera, and potato (see literature cited in Introduction). In order to determine if exon 1 of 5' rpl2 is intact in a few other core eudicots to which there was good hybridization on the blots, the genomic sequence of this region was obtained by PCR amplification from Gossypium, Ulmus, and Lonicera. Each sequenced region (ranging from 686–812 bp) was uninterrupted by stop codons or frameshifts.

3' rpl2 Has Been Transferred to the Nucleus in Four Angiosperm Families
To determine if 3' rpl2 has been transferred to the nucleus in core eudicots, searches of the NCBI sequence databases were performed. Nuclear 3' rpl2 cDNA sequences were discovered from tomato, cotton, Arabidopsis, soybean, and M. trunculata (see Materials and Methods for accession numbers). EST clones from Arabidopsis and soybean were purchased from Incyte Genomics and sequenced, whereas the available tomato and cotton EST sequences, and a contig of multiple Medicago ESTs, already represented full-length 3' rpl2 ORFs. The genomic sequence of Arabidopsis rpl2 is present on chromosome 2 (Lin et al. 1999Citation ), but it was not annotated as a gene, and rpl2 appears to be single copy in the mostly complete Arabidopsis genome sequence. An unpublished Arabidopsis 3' rpl2 cDNA sequence (X82556) is also available, but this sequence contains errors that caused the extent of the coding region to be incorrectly inferred relative to the data presented in this paper. The complete genomic sequences from cotton, tomato, and soybean were obtained by PCR amplification and sequencing, revealing one intron in each species (see below).

The nuclear 3' RPL2 aa sequences from each species have a well-conserved (relative to each other) region of about 155 aa that begins at a position corresponding to codon 354 in the entire RPL2 aa sequence from rice mitochondria (see fig. 5 ). The nuclear 3' RPL2 sequences include all of the carboxy terminal sequence that is well conserved among plants, protists, and bacteria. The mitochondrial rpl2-homologous region in the nuclear 3' rpl2 genes begins 32 codons downstream of the mitochondrial 5' rpl2 stop codon in eudicots (35 codons in rice); thus, a short segment that is not in a highly conserved region of the RPL2 protein seems to have been lost in the core eudicots. Each nuclear sequence contains a 5' extension of the ORF that has no similarity to the rice mitochondrial sequence. For each gene, a portion of this region is predicted by MITOPROT (Claros and Vincens 1996Citation ) and TargetP (Emanuelsson et al. 2000Citation ) to be a mitochondrial targeting presequence, although the predicted lengths vary. (Another mitochondrial protein prediction program—Predotar version 0.5 [www.inra.fr/Internet/Produits/Predotar]—predicts nuclear 3' rpl2 codes for a mitochondrial protein, but this program does not predict presequence cleavage sites.) The 5' extensions are not as well conserved among the different species as the corresponding mature coding regions, consistent with many other mitochondrially targeted proteins (reviewed in Glaser et al. 1998Citation ). Despite some length variation, the extensions align sufficiently well to be judged homologous (fig. 5 ). The genes from cotton and Arabidopsis contain a phase 1 intron that is in the same position; the genomic sequences from tomato and soybean also have an intron that appears to be in the same position, although this inference is partially dependent on sequence alignment (fig. 5 ). The evident homology (in both primary sequence and intron location) of the putative presequences of the nuclear 3' rpl2 genes strongly implies that they are the product of the same functional gene transfer event, an event that probably occurred early in eudicot evolution at about the same time as rpl2 truncation within the mitochondrial genome (see Discussion).



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Fig. 5.—Alignment of the 5' extensions of those nuclear rpl2 3' ORFs for which both genomic and cDNA sequences were determined. Identical aas are shown on a black background. Dashes indicate gaps inserted to improve alignment. Gray letters and the arrow indicate aas whose codons contain a phase 1 intron. Rectangle indicates the beginning of the mitochondrial rpl2-homologous region

 
The 5' rpl2 Gene Has Been Transferred to the Nucleus in Legumes
Considering that the 5' rpl2 ORF has been lost many times from the mitochondrial DNA in angiosperms (fig. 3 ), we searched the NCBI databases for the transferred nuclear copies. An intact nuclear 5' rpl2 ORF was discovered from the alignment of three soybean ESTs, and partial nuclear rpl2 EST sequences from Medicago and Lotus were also discovered (see Materials and Methods for accession numbers). The genomic sequence from the soybean was PCR-amplified and sequenced; it contains no introns. The soybean 5' rpl2 sequence is predicted by MITOPROT, TargetP, and Predotar version 0.5 to be mitochondrially targeted, but the sequence does not contain a 5' extension that might serve as a mitochondrial targeting presequence. Recent gene transfer and activation without gain of a presequence has been documented for one other ribosomal protein gene, rps10 (Adams et al. 2000Citation ; Kubo et al. 2000aCitation ). Soybean nuclear 5' rpl2 is 205 aa and ends at codon position 249 relative to the Oenothera mitochondrial 5' rpl2; thus the soybean gene product is 84 aa shorter than 5' rpl2 from Oenothera, with the missing parts being in regions that are not highly conserved. The 5' rpl2 gene in soybean and Medicago is not part of the same transcription unit as the 3' rpl2 gene in these species, and the genes are probably not linked. To sum up, both soybean and Medicago contain both parts of rpl2 in the nucleus, but as separate ORFs that were probably the result of different transfers (fig. 6 ).



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Fig. 6.—Structures of rpl2 in the mitochondrion and nucleus of various angiosperms. Genes located in the nucleus are shaded gray; presequences of nuclear genes are abbreviated as pre. Triangles indicate the locations of introns. The maize nuclear rpl2 represents a cDNA sequence. Sizes of various rpl2 coding regions include rice mitochondrial (503 aa), maize nuclear (449 aa), Arabidopsis 5' mitochondrial (349 aa), tomato 3' nuclear (203 aa), and soybean 5' nuclear (205 aa)

 
The Entire rpl2 Gene Has Been Transferred to the Nucleus in Maize
As 5' rpl2 has been lost from the mitochondrion of maize (fig. 3 ), we searched the NCBI EST databases to identify a transferred nuclear copy. Six ESTs were discovered that covered the 5' portion and 3' portion of rpl2, but not the middle portion. One EST clone was obtained from the maize ZmDB EST-sequencing project and fully sequenced. The maize nuclear rpl2 ORF corresponds in length to the full-length mitochondrial rpl2 in rice (i.e., the gene is not split as in core eudicots; see fig. 6 ). However, the maize nuclear RPL2 (449 aa) is shorter than the rice mitochondrial RPL2 (502 aa) because of a few deletions in the region corresponding to 5' RPL2 in the core eudicots, including a 33-bp deletion that spans the rpl2 fission site in core eudicots. MITOPROT and TargetP predict that the maize nuclear RPL2 contains a putative mitochondrial targeting presequence of 30–45 aa; however, alignment of the maize nuclear RPL2 with the mitochondrial RPL2 from rice shows only a 9-aa extension of the maize nuclear sequence. A partial rpl2 sequence (the 3' end) has been reported from the nucleus of wheat (Bonen, Carrillo, and Subramanian 1998Citation ), and it is likely that the wheat and maize rpl2 genes result from the same transfer.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Timing and Mechanism of rpl2 Fission and 3' rpl2 Transfer to the Nucleus
Two unusual phenomena have been presented in this study: the functional transfer of a portion of a mitochondrial gene to the nucleus and the fission of this gene in the mitochondrion. We now consider the phylogenetic timing of these events and their likely relationship.

We infer that 5' rpl2 was created from the entire rpl2 gene by the formation of a stop codon (TAA) in a common ancestor of core eudicots, after divergence of the Trochodendraceae (figs. 3 and 4 ). Three lines of evidence suggest that the 3' rpl2 nuclear genes from cotton, Arabidopsis, tomato, soybean, and Medicago are the result of a single transfer to the nucleus in a common ancestor of rosids and asterids. First, the mitochondrial targeting presequences of the nuclear 3' rpl2 gene from the four rosids and one asterid align relatively well and thus appear to be homologous (fig. 5 ). Second, the nuclear 3' rpl2 genes have an intron that is in the same position within the presequence (fig. 5 ). Third, the 3' rpl2 sequence does not appear ever to have been functional in core eudicots as a stand-alone ORF in the mitochondrial genome. Taken together, the three lines of evidence provide far stronger support for the hypothesis of a single transfer of the 3' region to the nucleus than for the hypothesis of multiple recent transfers. The functional transfer of the 3' region to the nucleus and the creation of the 5' ORF in the mitochondrion appear to have been approximately concurrent, given our current understanding of eudicot relationships (fig. 3 ).

Two models can be envisioned to explain rpl2 fission in the mitochondrion and transfer of the 3' region to the nucleus. In the first model, the entire rpl2 gene split into two functional ORFs, both initially resident in the mitochondrial genome. Soon afterwards, the 3' ORF was transferred to the nucleus. This was our hypothesis during the early stages of this project. However, as discussed earlier, there is no evidence that the 3' region was ever a functional ORF in core eudicot mitochondrial DNA. Thus, we do not favor the above model.

In the second, preferred model, the crucial initiating event was the functional transfer of the 3' region to the nucleus. This relieved selection on the corresponding 3' region of the ancestrally full-length rpl2 gene in the mitochondrion, allowing the mitochondrial 5' ORF to be formed via a point mutation that created a premature stop codon. In other words, rpl2 gene fission seems to be a consequence of partial gene transfer to the nucleus, although the two events were presumably independent. How could transfer and activation of only a portion of a mitochondrial gene in the nucleus occur? One possibility is that the entire gene was transferred to the nucleus, but only the 3' end became activated, perhaps because of a genomic rearrangement during presequence acquisition. Alternatively, the physical transfer event may have included only the 3' end of rpl2, either because of incomplete reverse transcription of mitochondrial rpl2 mRNA or because only a tiny fragment of the mitochondrial genome was transferred. The 3' region of rpl2 has only one RNA editing site in rice mitochondria and contains no introns (Kubo et al. 1996Citation ), raising the possibility of a DNA-mediated transfer of 3' rpl2 to the nucleus.

Under our preferred model, once 3' rpl2 became a functional gene in the nucleus, there was presumably a transition period when both the full-length mitochondrially synthesized RPL2 protein and the 3' RPL2 protein (synthesized in the cytosol) were present in the mitochondrion. Because the carboxy end of the full-length RPL2 protein was probably redundant to the 3' RPL2 protein, it is not clear if and how both proteins were assembled into the ribosome. Questions also arise as to how the mitochondrially synthesized 5' RPL2 protein and the cytosolically synthesized 3' RPL2 protein might assemble and interact in the ribosome. Do they in fact interact with one another in the ribosome (structurally, functionally, or both), and might they possibly even become fused together? Are they part of the same ribosomal protein assembly pathway, or are they assembled separately? Future biochemical experiments could address these questions.

Has there been a single transfer or multiple transfers of 5' rpl2 to the nucleus? The many, mostly recent and phylogenetically isolated losses of 5' rpl2 from core eudicot mitochondrial DNA, taken together with the presence of an intact 5' rpl2 exon 1 in the mitochondrion of all six core eudicots examined, suggest that there may have been many recent functional transfers of 5' rpl2. For one case of 5' rpl2 loss, in legumes, there is a corresponding gene in the nucleus that probably represents a recent transfer.

Mitochondrial Gene Fissions in Other Eukaryotes
Fission of genes in mitochondrial DNA appears to be a relatively rare process. Few examples are known, only one of which involves gene transfer to the nucleus, in addition to rpl2. The cytochrome c biogenesis gene that is homologous to the bacterial gene ccl1 is present as three separate ORFs in the liverwort Marchantia and in the Brassicaceae (with only one of the two fission points being shared), and as two separate ORFs in four other examined angiosperms (see Introduction). It appears that the ccl1 gene split once before the evolution of land plants, with the smaller portion (designated ccb452 in Arabidopsis) then splitting in the Marchantia lineage and the larger portion (ccb577) splitting in the Brassicaceae. Transfer of a cytochrome c biogenesis gene to the nucleus has not been reported in land plants. In another case of mitochondrial gene fission, the NADH dehydrogenase subunit 1 gene (nad1) has been split into two separately transcribed ORFs in Tetrahymena and Paramecium (Edqvist, Burger, and Gray 2000)Citation .

A particularly intriguing case of gene fission—one that does involve gene transfer to the nucleus—involves the cox2 gene in green algae. In Chlamydomonas and Polytomella, the cox2 gene is split into two separately expressed ORFs (cox2A and cox2B), and both ORFs are present in the nucleus (Pérez-Martínez et al. 2001Citation ). In another green alga, Prototheca, the entire cox2 gene is present in the mitochondrion as a single ORF (Wolff et al. 1994Citation ). Yet another green alga, Scenedesmus, contains a truncated cox2 gene (cox2A) in the mitochondrion (Kuck, Jekosch, and Holzamer 2000; Nedelcu et al. 2000)Citation , and it is hypothesized that the 3' end of cox2 (cox2B) is present in the nucleus of Scenedesmus (Pérez-Martínez et al. 2001Citation ). The variable presence of cox2 as one or two ORFs and in the nucleus or mitochondrion of green algae could be explained by two models. Pérez-Martínez et al. (2001)Citation proposed that there was cox2 fission in the mitochondrion, followed by transfers of each ORF (cox2A and cox2B) to the nucleus in the common ancestor of Polytomella and Chlamydomonas; presumably, the hypothesized nuclear cox2B in Scenedesmus would have been derived from a separate transfer. Alternatively, transfer of cox2B to the nucleus might have occurred first, in a common ancestor of Scenedesmus, Polytomella, and Chlamydomonas, followed by formation of the cox2A ORF in the mitochondrion, similar to what we have proposed for rpl2 in eudicots. In either model, cox2A was transferred to the nucleus in the lineage containing Polytomella and Chlamydomonas, after the divergence from the Scenedesmus lineage.

Perspectives on Gene Retention in the Mitochondrion Following Duplicative Transfer to the Nucleus
If our inference of a single transfer of the 3' end of rpl2 to the nucleus early during core eudicot evolution is correct, then numerous rosids and asterids have retained this region as a pseudogene in the mitochondrion for a relatively long period of time. Pseudogene retention on this scale is surprising, given the potential for deletion or recombination to eliminate the nonfunctional gene, and contrasts dramatically with the survey hybridization results obtained for the genes rps2 and rps11 in eudicots (Adams et al. 2001a). Rps11 and rps2 were probably transferred to the nucleus at about the same time as the 3' end of rpl2, yet the gene probes hybridized strongly to only one or two DNAs from the 179 core eudicots examined.

Why have pseudogenes of 3' rpl2 been so widely retained in eudicot mitochondrial DNAs? One possibility is that because the 3' end of rpl2 is tightly linked to the still-functional 5' rpl2 ORF, elimination by a large deletion is relatively difficult, as such a deletion might disrupt the 5' ORF. Other factors, such as the involvement of the 3' end in the regulation of the 5' ORF or in some other regulatory role, might also be at work. A very different possibility involves stochastic factors relating to the timing of mitochondrial gene loss relative to eudicot diversification. If 3' rpl2 happened to survive as a mitochondrial pseudogene through perhaps a rapid period of early eudicot diversification, then many separate mitochondrial losses, each occurring at the base of each of these early-arising lineages, would be required to produce an rps2/rps11–like hybridization pattern, and instead a pattern similar to that observed for 3' rpl2 might be more likely to result. Perhaps both rps2 and rps11 happened to be lost from the mitochondrial genome only once, relatively soon after transfer to the nucleus, before there was diversification into any of the many different descendant lineages of extant eudicots. (According to this model, the one or two deeply nested eudicot mitochondrial DNAs to which rps2 and rps11 probes hybridized strongly would have regained the relevant gene via reverse transfer from the nucleus or lateral transfer from some other taxa. Our preliminary data favor the latter possibility in the one case examined so far.) If so, then for rps2 or rps11, there would be no possibility of long-term retention of mitochondrial pseudogenes (and many separate losses within the eudicot transfer clade). Finally, it should be noted that these various selective and stochastic or phylogenetic possibilities are not mutually exclusive.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Yin-Long Qiu for providing the DNAs for the Southern blots and two anonymous reviewers for helpful comments on the manuscript. K.L.A. was supported by a U.S. Department of Agriculture Plant Biotechnology graduate fellowship and a William Ogg fellowship from Indiana University. Funding for this work came from National Institutes of Health grant GM-35087 to J.D.P.


    Footnotes
 
Elizabeth Kellogg, Reviewing Editor

Keywords: plant mitochondria gene fission gene transfer ribosomal protein genes Back

Abbreviations: aa, amino acids; ORF, open reading frame. Back

Address for correspondence and reprints: Keith L. Adams, Department of Botany, Iowa State University, Ames, Iowa 50011. kladams{at}iastate.edu . Back


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Accepted for publication August 27, 2001.