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Ribosomal protein L10 is encoded in the mitochondrial genome of many land plants and green algae

Identifieur interne : 000D99 ( Pmc/Corpus ); précédent : 000D98; suivant : 000E00

Ribosomal protein L10 is encoded in the mitochondrial genome of many land plants and green algae

Auteurs : Jeffrey P. Mower ; Linda Bonen

Source :

RBID : PMC:2780418

Abstract

Background

The mitochondrial genomes of plants generally encode 30-40 identified protein-coding genes and a large number of lineage-specific ORFs. The lack of wide conservation for most ORFs suggests they are unlikely to be functional. However, an ORF, termed orf-bryo1, was recently found to be conserved among bryophytes suggesting that it might indeed encode a functional mitochondrial protein.

Results

From a broad survey of land plants, we have found that the orf-bryo1 gene is also conserved in the mitochondria of vascular plants and charophycean green algae. This gene is actively transcribed and RNA edited in many flowering plants. Comparative sequence analysis and distribution of editing suggests that it encodes ribosomal protein L10 of the large subunit of the ribosome. In several lineages, such as crucifers and grasses, where the rpl10 gene has been lost from the mitochondrion, we suggest that a copy of the nucleus-encoded chloroplast-derived rpl10 gene may serve as a functional replacement.

Conclusion

Despite the fact that there are now over 20 mitochondrial genome sequences for land plants and green algae, this gene has remained unidentified and largely undetected until now because of the unlikely coincidence that most of the earlier sequences were from the few lineages that lack the intact gene. These results illustrate the power of comparative sequencing to identify novel genomic features.


Url:
DOI: 10.1186/1471-2148-9-265
PubMed: 19917118
PubMed Central: 2780418

Links to Exploration step

PMC:2780418

Le document en format XML

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<title>Background</title>
<p>The mitochondrial genomes of plants generally encode 30-40 identified protein-coding genes and a large number of lineage-specific ORFs. The lack of wide conservation for most ORFs suggests they are unlikely to be functional. However, an ORF, termed
<italic>orf-bryo1</italic>
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<title>Results</title>
<p>From a broad survey of land plants, we have found that the
<italic>orf-bryo1 </italic>
gene is also conserved in the mitochondria of vascular plants and charophycean green algae. This gene is actively transcribed and RNA edited in many flowering plants. Comparative sequence analysis and distribution of editing suggests that it encodes ribosomal protein L10 of the large subunit of the ribosome. In several lineages, such as crucifers and grasses, where the
<italic>rpl10 </italic>
gene has been lost from the mitochondrion, we suggest that a copy of the nucleus-encoded chloroplast-derived
<italic>rpl10 </italic>
gene may serve as a functional replacement.</p>
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<title>Conclusion</title>
<p>Despite the fact that there are now over 20 mitochondrial genome sequences for land plants and green algae, this gene has remained unidentified and largely undetected until now because of the unlikely coincidence that most of the earlier sequences were from the few lineages that lack the intact gene. These results illustrate the power of comparative sequencing to identify novel genomic features.</p>
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</TEI>
<pmc article-type="research-article">
<pmc-dir>properties open_access</pmc-dir>
<front>
<journal-meta>
<journal-id journal-id-type="nlm-ta">BMC Evol Biol</journal-id>
<journal-title-group>
<journal-title>BMC Evolutionary Biology</journal-title>
</journal-title-group>
<issn pub-type="epub">1471-2148</issn>
<publisher>
<publisher-name>BioMed Central</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="pmid">19917118</article-id>
<article-id pub-id-type="pmc">2780418</article-id>
<article-id pub-id-type="publisher-id">1471-2148-9-265</article-id>
<article-id pub-id-type="doi">10.1186/1471-2148-9-265</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Research article</subject>
</subj-group>
</article-categories>
<title-group>
<article-title>Ribosomal protein L10 is encoded in the mitochondrial genome of many land plants and green algae</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" corresp="yes" id="A1">
<name>
<surname>Mower</surname>
<given-names>Jeffrey P</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>jmower2@unl.edu</email>
</contrib>
<contrib contrib-type="author" id="A2">
<name>
<surname>Bonen</surname>
<given-names>Linda</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>lbonen@uOttawa.ca</email>
</contrib>
</contrib-group>
<aff id="I1">
<label>1</label>
Center for Plant Science Innovation and Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE, USA</aff>
<aff id="I2">
<label>2</label>
Department of Biology, University of Ottawa, Ottawa, Canada</aff>
<pub-date pub-type="collection">
<year>2009</year>
</pub-date>
<pub-date pub-type="epub">
<day>16</day>
<month>11</month>
<year>2009</year>
</pub-date>
<volume>9</volume>
<fpage>265</fpage>
<lpage>265</lpage>
<history>
<date date-type="received">
<day>3</day>
<month>8</month>
<year>2009</year>
</date>
<date date-type="accepted">
<day>16</day>
<month>11</month>
<year>2009</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright ©2009 Mower and Bonen; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2009</copyright-year>
<copyright-holder>Mower and Bonen; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0">
<license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<self-uri xlink:href="http://www.biomedcentral.com/1471-2148/9/265"></self-uri>
<abstract>
<sec>
<title>Background</title>
<p>The mitochondrial genomes of plants generally encode 30-40 identified protein-coding genes and a large number of lineage-specific ORFs. The lack of wide conservation for most ORFs suggests they are unlikely to be functional. However, an ORF, termed
<italic>orf-bryo1</italic>
, was recently found to be conserved among bryophytes suggesting that it might indeed encode a functional mitochondrial protein.</p>
</sec>
<sec>
<title>Results</title>
<p>From a broad survey of land plants, we have found that the
<italic>orf-bryo1 </italic>
gene is also conserved in the mitochondria of vascular plants and charophycean green algae. This gene is actively transcribed and RNA edited in many flowering plants. Comparative sequence analysis and distribution of editing suggests that it encodes ribosomal protein L10 of the large subunit of the ribosome. In several lineages, such as crucifers and grasses, where the
<italic>rpl10 </italic>
gene has been lost from the mitochondrion, we suggest that a copy of the nucleus-encoded chloroplast-derived
<italic>rpl10 </italic>
gene may serve as a functional replacement.</p>
</sec>
<sec>
<title>Conclusion</title>
<p>Despite the fact that there are now over 20 mitochondrial genome sequences for land plants and green algae, this gene has remained unidentified and largely undetected until now because of the unlikely coincidence that most of the earlier sequences were from the few lineages that lack the intact gene. These results illustrate the power of comparative sequencing to identify novel genomic features.</p>
</sec>
</abstract>
</article-meta>
</front>
<body>
<sec>
<title>Background</title>
<p>The mitochondrial proteome consists of at least 1000 different proteins. The genes encoding many of these proteins were initially encoded within the original respiring endosymbiont but have undergone intracellular transfer to the nucleus over evolutionary time, so that the proteins must be targeted back to the mitochondrion to perform their function. The number of retained mitochondrial protein-coding genes varies widely among eukaryotes, from 67 in the jakobid
<italic>Reclinomonas americana </italic>
[
<xref ref-type="bibr" rid="B1">1</xref>
] to only 3 in apicomplexans such as
<italic>Plasmodium falciparum </italic>
[
<xref ref-type="bibr" rid="B2">2</xref>
]. Genes retained in the mitochondrion encode proteins involved in fundamental mitochondrial processes such as electron transport, ATP synthesis, gene expression, and protein maturation/import. In
<italic>Reclinomonas </italic>
mitochondria, genes for the translational machinery comprise the largest single category, with 27 ribosomal protein genes [
<xref ref-type="bibr" rid="B1">1</xref>
].</p>
<p>In streptophytes (vascular plants, bryophytes, and charophycean green algae), the mitochondrial genome typically contains about 30 to 40 protein-coding genes of identified function. Approximately 20 of these genes are universally present, whereas the others (or a subset thereof) have been lost from various plant groups [
<xref ref-type="bibr" rid="B3">3</xref>
]. Genes encoding ribosomal proteins and subunits of the succinate dehydrogenase complex are most commonly absent [
<xref ref-type="bibr" rid="B3">3</xref>
], although loss or pseudogenization of other genes, such as
<italic>cox2 </italic>
[
<xref ref-type="bibr" rid="B4">4</xref>
,
<xref ref-type="bibr" rid="B5">5</xref>
],
<italic>nad7 </italic>
[
<xref ref-type="bibr" rid="B6">6</xref>
,
<xref ref-type="bibr" rid="B7">7</xref>
],
<italic>atp8 </italic>
[
<xref ref-type="bibr" rid="B7">7</xref>
], and cytochrome c biogenesis subunits [
<xref ref-type="bibr" rid="B7">7</xref>
,
<xref ref-type="bibr" rid="B8">8</xref>
] has occurred as well. Typically, a gene is deleted from the plant mitochondrial genome only after successful transfer of a copy to the nucleus, although examples exist where loss is correlated with functional replacement of a "native" mitochondrial ribosomal protein by a nucleus-encoded plastid or cytosolic homolog [
<xref ref-type="bibr" rid="B9">9</xref>
,
<xref ref-type="bibr" rid="B10">10</xref>
]. The timing of migration of mitochondrial ribosomal protein genes to the nucleus during eukaryotic evolution can be followed by comparative analysis [
<xref ref-type="bibr" rid="B11">11</xref>
,
<xref ref-type="bibr" rid="B12">12</xref>
].</p>
<p>The mitochondrial genomes of seed plants are particularly large and recombinogenic. They contain many potential unknown open reading frames (ORFs) which have often been annotated as such in genomic sequencing projects when longer than 100 codons. However, most of these ORFs are not broadly conserved, which has brought into question their potential functionality. Moreover, it is not uncommon for plant mitochondrial DNA rearrangements to give rise to novel chimeric ORFs in specific lineages, and in certain instances such ORFs are correlated with mitochondrial dysfunction in the form of cytoplasmic male sterility [
<xref ref-type="bibr" rid="B13">13</xref>
]. On the other hand, a few ORFs have shown conservation among plants, and over recent years these have been upgraded to known mitochondrial genes. This list includes
<italic>atp4 </italic>
[
<xref ref-type="bibr" rid="B14">14</xref>
,
<xref ref-type="bibr" rid="B15">15</xref>
],
<italic>atp8 </italic>
[
<xref ref-type="bibr" rid="B15">15</xref>
-
<xref ref-type="bibr" rid="B17">17</xref>
] and
<italic>mttB </italic>
(or
<italic>tatC</italic>
) [
<xref ref-type="bibr" rid="B18">18</xref>
,
<xref ref-type="bibr" rid="B19">19</xref>
], which previously were denoted as
<italic>orf25</italic>
,
<italic>orfB</italic>
, and
<italic>orfX</italic>
, respectively. Within the three complete non-vascular plant mitochondrial genomes, there is another unidentified conserved ORF, named
<italic>orf-bryo1 </italic>
in the hornwort
<italic>Megaceros aenigmaticus </italic>
[
<xref ref-type="bibr" rid="B7">7</xref>
],
<italic>orf187 </italic>
in the moss
<italic>Physcomitrella patens </italic>
[
<xref ref-type="bibr" rid="B20">20</xref>
], and
<italic>orf168 </italic>
in the liverwort
<italic>Marchantia polymorpha </italic>
[
<xref ref-type="bibr" rid="B21">21</xref>
], suggesting that it may in fact code for a functional mitochondrial product in plants.</p>
</sec>
<sec>
<title>Results and Discussion</title>
<sec>
<title>Mitochondrial
<italic>orf-bryo1 </italic>
is conserved across streptophytes</title>
<p>To determine whether this bryophyte mitochondrial ORF might be more widespread among plants, blastp searches were performed using these three protein sequences to query the NCBI protein database. A homolog was found in the completely sequenced mitochondrial genomes of the angiosperms
<italic>Nicotiana tabacum </italic>
(
<italic>orf159b</italic>
) [
<xref ref-type="bibr" rid="B22">22</xref>
] and
<italic>Vitis vinifera </italic>
(
<italic>orf159</italic>
) [
<xref ref-type="bibr" rid="B23">23</xref>
] and, albeit with low sequence similarity, in the charophytes
<italic>Chaetosphaeridium globosum </italic>
(
<italic>orf126</italic>
) [
<xref ref-type="bibr" rid="B8">8</xref>
] and
<italic>Chlorokybus atmophyticus </italic>
(
<italic>orf295</italic>
) [
<xref ref-type="bibr" rid="B24">24</xref>
]. An unnamed predicted protein from cDNA analysis (
<ext-link ext-link-type="gen" xlink:href="XP_002332837">XP_002332837</ext-link>
) was also identified from
<italic>Populus trichocarpa</italic>
. Interestingly, the moss
<italic>orf187 </italic>
shows weak similarity to ribosomal protein L10 from several bacteria, including
<italic>Rickettsia prowazekii </italic>
and other members of the alpha-proteobacteria, the lineage from which mitochondria originated [
<xref ref-type="bibr" rid="B25">25</xref>
], as well as to mitochondrial L10 from the jakobid
<italic>Reclinomonas americana</italic>
, a protist that possesses the most "primitive" and gene-rich of all mitochondrial genomes [
<xref ref-type="bibr" rid="B1">1</xref>
]. These observations suggested that the moss
<italic>orf187 </italic>
(and its homologs) might encode mitochondrial L10 in plants. Indeed, annotated L10 domains can be found in the GenPept records for
<italic>Physcomitrella orf187 </italic>
(
<ext-link ext-link-type="gen" xlink:href="BAE93086">BAE93086</ext-link>
) and
<italic>Chlorokybus orf295 </italic>
(
<ext-link ext-link-type="gen" xlink:href="ABO15139">ABO15139</ext-link>
).</p>
<p>A variety of computational and experimental approaches were used to determine the distribution of mitochondrial
<italic>rpl10</italic>
-like sequences among streptophytes, and the results are summarized in Figure
<xref ref-type="fig" rid="F1">1</xref>
. To extend the database search, tblastn queries were conducted against the nucleotide nr and EST-others databases at GenBank. Indeed, homologous unannotated ORFs are present within the complete mitochondrial genomes of the charophyte
<italic>Chara vulgaris</italic>
, [
<xref ref-type="bibr" rid="B26">26</xref>
], the gymnosperm
<italic>Cycas taitungensis </italic>
[
<xref ref-type="bibr" rid="B27">27</xref>
], and the angiosperm
<italic>Carica papaya </italic>
(
<ext-link ext-link-type="gen" xlink:href="EU431224">EU431224</ext-link>
) as well as in partial mitochondrial genome entries for the angiosperms
<italic>Solanum lycopersicum </italic>
and
<italic>Helianthus annuus</italic>
. In addition, several truncated and/or frameshifted sequences were identified in the mitochondrial genomes of
<italic>Brassica napus, Oryza sativa</italic>
, and
<italic>Bambusa oldhamii</italic>
, suggestive of recent erosion of the
<italic>rpl10</italic>
-like gene. Searches of the EST-others database also revealed numerous homologs from a wide range of angiosperms as well as two gymnosperms,
<italic>Picea glauca </italic>
and
<italic>Welwitschia mirabilis</italic>
. Their high nucleotide similarity to counterparts identified in completely sequenced mitochondrial genomes of other seed plants suggests that these are in fact encoded in the mitochondrial genome, unless there has been extremely recent gene transfer to the nucleus. One exception is a divergent
<italic>rpl10</italic>
-like sequence from the fern
<italic>Adiantum capillus-veneris </italic>
(
<ext-link ext-link-type="gen" xlink:href="DK949045">DK949045</ext-link>
) that has an amino-terminal extension of 25 residues with a weak predicted mitochondrial targeting signal, and might therefore be nuclear-located.</p>
<fig id="F1" position="float">
<label>Figure 1</label>
<caption>
<p>
<bold>Distribution of mitochondrial
<italic>rpl10</italic>
-like sequences in streptophytes</bold>
. Functional genes, pseudogenes, and genes lost from the mitochondrion are shown as filled squares, open squares, and open circles, respectively. Genes with evidence for expression as determined by RNA editing status are marked with a plus symbol. The 'nuc?' note next to the
<italic>Adiantum </italic>
sequence indicates that it may be encoded in the nucleus. The origin of each sequence is given in parentheses using the following abbreviations: E - EST sequence from GenBank; G - genome sequence from GenBank; N - nucleotide sequence from GenBank; P - PCR product generated during this study; R - RT-PCR product generated during this study. Phylogenetic relationships are taken from the Angiosperm Phylogeny Website [
<xref ref-type="bibr" rid="B55">55</xref>
].</p>
</caption>
<graphic xlink:href="1471-2148-9-265-1"></graphic>
</fig>
<p>To determine how widely this mitochondrial
<italic>rpl10</italic>
-like gene is represented in seed plants and to gain more insight into the prevalence and timing of apparent pseudogenization in certain lineages, a PCR survey was undertaken using primers designed from the angiosperm and gymnosperm sequences identified above. Sequencing revealed the presence of this gene in another 24 seed plants, of which 5 were pseudogenes (Figure
<xref ref-type="fig" rid="F1">1</xref>
). Overall, these results show that homologs to the
<italic>orf-bryo1 </italic>
gene can be found across virtually all major streptophyte lineages, although it should be noted that lycophytes are not represented in this data set and no homologous sequences were detected in the mitochondrial data recently presented for
<italic>Isoetes engelmannii </italic>
[
<xref ref-type="bibr" rid="B28">28</xref>
]. Notably, the
<italic>rpl10</italic>
-like gene appears to have been independently lost at least five times during angiosperm history: from the asterid
<italic>Pentas</italic>
, from the caryophyllid
<italic>Beta</italic>
, from the crucifers
<italic>Arabidopsis </italic>
and
<italic>Brassica</italic>
, from monocots, and from the conifer
<italic>Podocarpus</italic>
.</p>
</sec>
<sec>
<title>Angiosperm
<italic>orf-bryo1 </italic>
homologs are transcribed, edited and likely encode a functional mitochondrial L10</title>
<p>At the DNA level, the mitochondrial
<italic>rpl10</italic>
-like gene appears to be functional in a very wide range of streptophytes, and the derived amino acid sequence alignments for selected species are shown in Figure
<xref ref-type="fig" rid="F2">2</xref>
. Amino acid conservation is higher in the amino-terminal region than at the carboxy-terminus, and the latter also shows variation in length, in keeping with features also common to L10 proteins in non-plants (see below). The initiation codons for
<italic>Cycas </italic>
and
<italic>Megaceros </italic>
are predicted to be generated by C-to-U RNA editing of ACG to AUG. Within the
<italic>Megaceros </italic>
coding sequence, three potential stop codons are presumably removed by U-to-C RNA editing prior to translation, as previously postulated for many
<italic>Megaceros </italic>
mitochondrial transcripts including
<italic>orf-bryo1 </italic>
[
<xref ref-type="bibr" rid="B7">7</xref>
]. To assess whether the coding sequences are under functional constraint, the ratio (
<italic>ω</italic>
) of non-synonymous (
<italic>d</italic>
<sub>N</sub>
) to synonymous (
<italic>d</italic>
<sub>S</sub>
) divergence was calculated for all pairwise sequence comparisons between 6 representative streptophytes (Table
<xref ref-type="table" rid="T1">1</xref>
). In all 15 cases,
<italic>ω </italic>
was less than 1 consistent with purifying selection acting to maintain the protein sequences. The average over all tests was 0.39 with a high of 0.62 between
<italic>Marchantia </italic>
and
<italic>Cycas </italic>
and a low of 0.19 between
<italic>Chara </italic>
and
<italic>Cycas</italic>
.</p>
<fig id="F2" position="float">
<label>Figure 2</label>
<caption>
<p>
<bold>Alignment of L10 ribosomal proteins from plant mitochondria and eubacteria</bold>
. Amino acids within a column are shaded if at least 75% are identical (black) or similar (gray). Columns in which RNA editing was observed in one or more sequences are marked with a red asterisk, and those positions are shaded in red. In the sequences translated from DNA, positions shown in lowercase and shaded in yellow were inferred to result from RNA editing by comparison to sequences from
<italic>Physcomitrella, Marchantia, and Chara </italic>
and from angiosperms with known editing data.</p>
</caption>
<graphic xlink:href="1471-2148-9-265-2"></graphic>
</fig>
<table-wrap id="T1" position="float">
<label>Table 1</label>
<caption>
<p>Pairwise ω (
<italic>d</italic>
<sub>N</sub>
/
<italic>d</italic>
<sub>S</sub>
) for plant
<italic>rpl10 </italic>
sequences</p>
</caption>
<table frame="hsides" rules="groups">
<tbody>
<tr>
<td align="left">
<italic>Petunia</italic>
</td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Citrus</italic>
</td>
<td align="left">0.414</td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Cycas</italic>
</td>
<td align="left">0.517</td>
<td align="left">0.557</td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Physcomitrella</italic>
</td>
<td align="left">0.345</td>
<td align="left">0.423</td>
<td align="left">0.327</td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Marchantia</italic>
</td>
<td align="left">0.476</td>
<td align="left">0.504</td>
<td align="left">0.620</td>
<td align="left">0.476</td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Chara</italic>
</td>
<td align="left">0.222</td>
<td align="left">0.256</td>
<td align="left">0.181</td>
<td align="left">0.256</td>
<td align="left">0.332</td>
<td></td>
</tr>
<tr>
<td></td>
<td colspan="6">
<hr></hr>
</td>
</tr>
<tr>
<td></td>
<td align="left">
<italic>Pet.</italic>
</td>
<td align="left">
<italic>Cit.</italic>
</td>
<td align="left">
<italic>Cyc.</italic>
</td>
<td align="left">
<italic>Phy.</italic>
</td>
<td align="left">
<italic>Mar.</italic>
</td>
<td align="left">
<italic>Cha.</italic>
</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>We have also established that the mitochondrial
<italic>rpl10</italic>
-like gene is expressed and edited in angiosperms (Table
<xref ref-type="table" rid="T2">2</xref>
). The cDNA sequences obtained from four angiosperm species (
<italic>Aristolochia</italic>
,
<italic>Artemisia</italic>
,
<italic>Breynia </italic>
and
<italic>Ceropegia</italic>
) all showed C-to-U RNA editing at between 5 and 8 sites, which verifies that they were derived from RNA template rather than contaminating mitochondrial DNA. In addition, 7 edit sites were identified for
<italic>Citrus </italic>
by comparison of its EST and gene sequences. Editing in all 5 plants predominantly alters the encoded amino acids, with each coding sequence having only one silent editing event. Furthermore, these non-synonymous editing events improve protein similarity of the angiosperm sequences to one another and to species that are known to have infrequent editing, such as
<italic>Physcomitrella </italic>
[
<xref ref-type="bibr" rid="B29">29</xref>
], or no editing, as for
<italic>Marchantia </italic>
[
<xref ref-type="bibr" rid="B21">21</xref>
] and
<italic>Chara </italic>
[
<xref ref-type="bibr" rid="B26">26</xref>
]. This pattern of editing is characteristic for functional plant mitochondrial genes but not necessarily for pseudogenes [
<xref ref-type="bibr" rid="B30">30</xref>
], and most unconserved ORFs are not edited at all [
<xref ref-type="bibr" rid="B31">31</xref>
-
<xref ref-type="bibr" rid="B33">33</xref>
]. The
<italic>rpl10</italic>
-like EST sequences provided further evidence of transcription, although in the absence of accompanying DNA sequence information the evidence is less certain. The EST sequences from
<italic>Petunia, Theobroma</italic>
, and
<italic>Zinnia </italic>
generally have T at confirmed edit positions and therefore likely derive from genuine RNA rather than mitochondrial DNA contamination, although it cannot be excluded that some of these T residues are already encoded in the genome. In contrast, homologs based on EST data from additional plants lack several expected edits and reflect either mitochondrial DNA contamination or partially edited transcripts (data not shown). In total,
<italic>rpl10</italic>
-like sequences from 8 distantly-related angiosperms provide strong evidence for appropriate expression at the RNA level (Figure
<xref ref-type="fig" rid="F1">1</xref>
).</p>
<table-wrap id="T2" position="float">
<label>Table 2</label>
<caption>
<p>The effect of RNA editing on amino acid sequence</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th></th>
<th></th>
<th align="center" colspan="8">Confirmed Nonsilent Editing Positions
<sup>a</sup>
</th>
</tr>
<tr>
<th></th>
<th></th>
<th colspan="8">
<hr></hr>
</th>
</tr>
<tr>
<th align="left">Species</th>
<th align="left">Evidence</th>
<th align="right">31</th>
<th align="right">33</th>
<th align="right">39</th>
<th align="right">52</th>
<th align="right">73</th>
<th align="right">109</th>
<th align="right">156</th>
<th align="right">189</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left">
<italic>Aristolochia</italic>
</td>
<td align="left">DNA+cDNA</td>
<td align="right">P>L</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">P>L</td>
<td align="right">P>L</td>
<td align="right">S>L</td>
<td align="right">S>L</td>
<td align="right">L>F</td>
</tr>
<tr>
<td align="left">
<italic>Artemisia</italic>
</td>
<td align="left">DNA+cDNA</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">S>L</td>
<td align="right">P>L</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">L</td>
<td align="right">L</td>
</tr>
<tr>
<td align="left">
<italic>Breynia</italic>
<sup>b</sup>
</td>
<td align="left">DNA+cDNA</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">S>L</td>
<td align="right">P>L</td>
<td align="right">P>L</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">L</td>
</tr>
<tr>
<td align="left">
<italic>Ceropegia</italic>
<sup>b</sup>
</td>
<td align="left">DNA+cDNA</td>
<td align="right">L</td>
<td align="right">
<bold>
<italic>S</italic>
</bold>
</td>
<td align="right">S>L</td>
<td align="right">P>L</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">L</td>
<td align="right">F</td>
</tr>
<tr>
<td align="left">
<italic>Citrus</italic>
</td>
<td align="left">DNA+EST</td>
<td align="right">L</td>
<td align="right">S>L</td>
<td align="right">S>L</td>
<td align="right">S>L</td>
<td align="right">P>L</td>
<td align="right">S>L</td>
<td align="right">S>L</td>
<td align="right">L</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Petunia</italic>
</td>
<td align="left">EST</td>
<td align="right">L</td>
<td align="right">
<bold>
<italic>S</italic>
</bold>
</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
</tr>
<tr>
<td align="left">
<italic>Theobroma</italic>
</td>
<td align="left">EST</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
</tr>
<tr>
<td align="left">
<italic>Zinnia</italic>
</td>
<td align="left">EST</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">
<bold>
<italic>S</italic>
</bold>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">
<italic>Cycas</italic>
</td>
<td align="left">DNA</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">I</td>
</tr>
<tr>
<td align="left">
<italic>Physcomitrella</italic>
</td>
<td align="left">DNA</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">F</td>
</tr>
<tr>
<td align="left">
<italic>Marchantia</italic>
</td>
<td align="left">DNA</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">I</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">F</td>
</tr>
<tr>
<td align="left">
<italic>Chara</italic>
</td>
<td align="left">DNA</td>
<td align="right">I</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">L</td>
<td align="right">F</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>a: Non-conserved amino acids are shown in bold italic type.</p>
<p>b: Two editing events occur at codon position 52 for
<italic>Breynia </italic>
and
<italic>Ceropegia</italic>
.</p>
</table-wrap-foot>
</table-wrap>
<p>In Figure
<xref ref-type="fig" rid="F2">2</xref>
, the amino acid alignment of plant and charophycean green algal mitochondrial
<italic>orf-bryo1 </italic>
homologs also includes the
<italic>Reclinomonas americana </italic>
mitochondrion-encoded L10 protein and homologs from the eubacteria
<italic>Escherichia coli</italic>
,
<italic>Rickettsia prowazekii</italic>
, and
<italic>Thermotoga maritima</italic>
. The L10 ribosomal protein is universally present in the ribosomes of eubacteria, archaea, and eukaryotes, and the crystal structure of L10-L7/L12 stalk has been determined [
<xref ref-type="bibr" rid="B34">34</xref>
]. It is worth noting that the amino-terminal domain is more highly conserved than the carboxy-terminal half. For example, the
<italic>Rickettsia prowazekii </italic>
and
<italic>E. coli </italic>
L10 proteins share only about 26% amino acid identity over their full length, whereas the beta-1 to alpha-5 region (of 85 amino acids) within the amino-terminal half shows ~35% identity. It is the amino-terminal domain of L10 (or more specifically, the alpha-1 to alpha-3 region) that binds directly to the large subunit ribosomal RNA, whereas the carboxy-terminal domain of L10 (and alpha-8 in particular) interacts with the L7/L12 stalk; together with L11, this complex plays a key role in recruiting translation factors to the ribosome and stimulating GTP hydrolysis [
<xref ref-type="bibr" rid="B34">34</xref>
,
<xref ref-type="bibr" rid="B35">35</xref>
]. The flowering plant mitochondrial L10 proteins share about 23% amino acid identity with the
<italic>Rickettsia </italic>
L10 homolog over the amino-terminal beta-1 to alpha-5 region of 85 amino acids, compared to 27-28% identity seen between
<italic>Rickettsia </italic>
L10 and the comparable region of the
<italic>Physcomitrella </italic>
or
<italic>Reclinomonas </italic>
mitochondrial counterparts. Of particular note are several highly conserved blocks that are believed to be important for protein structure [
<xref ref-type="bibr" rid="B34">34</xref>
]. They contain Gly (and Pro) residues for beta-turns between beta1-alpha2 and alpha4-beta3 in L10 proteins of eubacteria and archaea. Interestingly, 7 of 8 positions of RNA editing lie within conserved blocks, consistent with their functional importance, a hallmark of RNA editing in plant mitochondria [
<xref ref-type="bibr" rid="B36">36</xref>
].</p>
<p>In bacterial, archaeal and eukaryotic cytosolic ribosomes, the amino terminal domain of the L10 protein is known to bind specifically to helices H42, H43, and H44 of the large subunit rRNA [
<xref ref-type="bibr" rid="B34">34</xref>
,
<xref ref-type="bibr" rid="B35">35</xref>
], and in plant mitochondria, this helical region of the 26S rRNA has retained the correct structure for L10 binding and is very highly conserved among streptophytes (Figure
<xref ref-type="fig" rid="F3">3</xref>
). Indeed this stretch of 80 nt is identical in sequence among most seed plants and there has been only one nucleotide substitution relative to either the
<italic>Physcomitrella </italic>
or
<italic>Marchantia </italic>
homolog, that is, during a period of about 400 million years. Thus, it seems likely that a conventional L10 protein (or at least for the amino-terminal portion) will be present in plant mitochondrial ribosomes.</p>
<fig id="F3" position="float">
<label>Figure 3</label>
<caption>
<p>
<bold>Sequence and structure of the LSU rRNA region that binds to L10 ribosomal protein</bold>
. Shown are helices H42, H43 and H44 of the LSU rRNA. The primary sequence shown is a consensus of this mitochondrial 26S rRNA region from
<italic>Marchantia, Physcomitrella</italic>
, and numerous seed plants, with differences shown in red. Positions that differ between plant mitochondria and bacteria (represented by
<italic>E. coli</italic>
) are shaded in gray. Yellow shading indicates compensatory changes in
<italic>E. coli </italic>
that maintain base pairing in stem regions. Nucleotide coordinates are shown for
<italic>Triticum aestivum </italic>
mitochondrial 26s rRNA [
<xref ref-type="bibr" rid="B56">56</xref>
] and in parentheses for
<italic>E. coli </italic>
23s rRNA [
<xref ref-type="bibr" rid="B34">34</xref>
,
<xref ref-type="bibr" rid="B35">35</xref>
].</p>
</caption>
<graphic xlink:href="1471-2148-9-265-3"></graphic>
</fig>
</sec>
<sec>
<title>Status of mitochondrial L10 in grasses and crucifers</title>
<p>For the reasons discussed above, one might expect that all seed plants would possess a mitochondrial-type
<italic>rpl10 </italic>
gene either within the mitochondrion or alternatively within the nucleus since the simplest explanation for cases of gene loss from the mitochondrion (see Figure
<xref ref-type="fig" rid="F1">1</xref>
) is that successful gene transfer to the nucleus has occurred. Curiously, no mitochondrial-type L10 protein sequences were detected in tblastn searches of the completely sequenced nuclear genomes of Arabidopsis [
<xref ref-type="bibr" rid="B37">37</xref>
] or rice [
<xref ref-type="bibr" rid="B38">38</xref>
,
<xref ref-type="bibr" rid="B39">39</xref>
]. However, both these genomes do contain duplicated copies of the chloroplast-derived
<italic>rpl10 </italic>
gene (data not shown). In land plants, the chloroplast
<italic>rpl10 </italic>
gene is located in the nucleus, and proteomic analysis of spinach chloroplast ribosomes has established its precise protein content [
<xref ref-type="bibr" rid="B40">40</xref>
]. The chloroplast L10 orthologs in Arabidopsis (
<ext-link ext-link-type="gen" xlink:href="NP_196855">NP_196855</ext-link>
) and rice (
<ext-link ext-link-type="gen" xlink:href="NP_001049761">NP_001049761</ext-link>
) share about 70% amino acid identity (excluding the acquired N-terminal targeting extensions). In contrast, the second chloroplast-type L10-related copy shows only ~41% amino acid identity between the Arabidopsis (
<ext-link ext-link-type="gen" xlink:href="NP_187843">NP_187843</ext-link>
) and rice (
<ext-link ext-link-type="gen" xlink:href="NP_001054498">NP_001054498</ext-link>
) counterparts, and these proteins are predicted to be localized in the mitochondrion based on targeting programs such as TargetP [
<xref ref-type="bibr" rid="B41">41</xref>
], PSort [
<xref ref-type="bibr" rid="B42">42</xref>
], and Predotar [
<xref ref-type="bibr" rid="B43">43</xref>
] Interestingly, the two Arabidopsis chloroplast-derived L10 paralogs are more closely related to each other (~58% identity) than are two rice ones (~46% identity), suggesting a more recent duplication event in the crucifer lineage. This would also be consistent with their independent recruitment as functional substitutes for the mitochondrial L10 protein at different times during angiosperm evolution, although it cannot be formally excluded that gene conversion events in the Arabidopsis lineage contribute to the higher sequence similarity.</p>
<p>Although the duplicated chloroplast-type L10-related gene is an attractive candidate to serve as a replacement in the mitochondrial ribosome for those plants which lack the "native" mitochondrial
<italic>rpl10 </italic>
gene, these proteins in Arabidopsis and rice lack a number of the expected conserved residues, ones that are observed in the plant mitochondrion-encoded genes. Alternative possibilities are that the chloroplast L10 might be dual targeted to both the plastid and the mitochondrion or that the cytosolic ribosomal protein L10 counterpart (called L10e or P0) has been recruited. It is perhaps even possible that plants such as rice and
<italic>Brassica</italic>
, which possess what appear to be remnant pseudogene fragments in the mitochondrion, actually have several short genes (mitochondrial or nuclear) that generate a discontinuous L10 protein structure, a phenomenon observed for the mitochondrial
<italic>rpl2 </italic>
gene in certain flowering plants [
<xref ref-type="bibr" rid="B44">44</xref>
]. Finally, it is worth noting that non-homologous proteins have been known to perform molecular mimicry in the evolution of the large ribosomal subunit among eubacteria and archaea [
<xref ref-type="bibr" rid="B45">45</xref>
].</p>
</sec>
</sec>
<sec>
<title>Conclusion</title>
<p>In summary, these observations provide strong evidence that a functional
<italic>rpl10 </italic>
gene exists in the mitochondrion of many streptophytes. Despite the fact that there are now over 20 streptophytes with complete mitochondrial genome sequences, this gene has been missed until now due to the unlikely coincidence that most of the plant mitochondrial genomes that were first completely sequenced - the crucifers
<italic>Arabidopsis thaliana </italic>
[
<xref ref-type="bibr" rid="B46">46</xref>
] and
<italic>Brassica napus </italic>
[
<xref ref-type="bibr" rid="B33">33</xref>
]; the grasses
<italic>Oryza sativa </italic>
[
<xref ref-type="bibr" rid="B32">32</xref>
],
<italic>Zea mays </italic>
[
<xref ref-type="bibr" rid="B47">47</xref>
] and
<italic>Triticum aestivum </italic>
[
<xref ref-type="bibr" rid="B48">48</xref>
]; and the sugar beet
<italic>Beta vulgaris </italic>
[
<xref ref-type="bibr" rid="B49">49</xref>
] - are from lineages where this gene has been lost or pseudogenized. Only with the more recent sequence data from diverse streptophytes such as
<italic>Cycas taitungensis </italic>
[
<xref ref-type="bibr" rid="B27">27</xref>
],
<italic>Physcomitrella patens </italic>
[
<xref ref-type="bibr" rid="B20">20</xref>
] and
<italic>Megaceros aenigmaticus </italic>
[
<xref ref-type="bibr" rid="B7">7</xref>
] does the general pattern emerge that this gene is in fact widely present. Indeed, the bryophyte
<italic>orf-bryo1 </italic>
sequences were particularly informative in bridging the evolutionary distance between mitochondrial L10 gene homologs in seed plants and those of charophycean green algae/protists, which nicely illustrates the power of obtaining sequence information from diverse organisms in order to reconstruct events related to gene and genome history.</p>
</sec>
<sec sec-type="methods">
<title>Methods</title>
<p>Total genomic DNAs and RNAs were isolated using the DNeasy and RNeasy Plant Mini Kits (QIAGEN) from leaf tissue available in the living collection of the Beadle Center Greenhouse (University of Nebraska). To prepare first-strand cDNA, RNAs were treated with DNase I (Fermentas) to remove contaminating DNA and then reverse transcribed using M-MuLV Reverse Transcriptase (Fermentas) and random hexamers (Fermentas) according to the manufacturer's instructions.</p>
<p>Sequences for
<italic>rpl10 </italic>
were amplified from DNA or cDNA by polymerase chain reaction using GoTaq DNA Polymerase (Promega) and forward primer F1 (5'-ATGCCATTCGGAAGAAGTMT) with reverse primer R159 (5'-TTAGGTGGTATYCCGAGATYGA) or R148 (5'-GGAACACACGAAASAAAGATATRAAC). Each reaction was run on a DNA Engine Dyad (Bio-Rad) for 35 cycles (30 sec at 94°C, 1 min at 48°C, 2 min at 72°C), with an initial step of 3 min at 94°C and a final step of 10 min at 72°C. Amplified products were sequenced on both strands at the High-Throughput Genomics Unit (University of Washington). Sequences generated in this study were deposited in GenBank under accession numbers
<ext-link ext-link-type="gen" xlink:href="GQ402491">GQ402491</ext-link>
-
<ext-link ext-link-type="gen" xlink:href="GQ402514">GQ402514</ext-link>
; additional sequences used in the comparative analysis were downloaded from GenBank (Table
<xref ref-type="table" rid="T3">3</xref>
).</p>
<table-wrap id="T3" position="float">
<label>Table 3</label>
<caption>
<p>Taxonomy and GenBank accession numbers for
<italic>rpl10 </italic>
sequences in this study</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th align="left">Group</th>
<th align="left">Order</th>
<th align="left">Species</th>
<th align="left">Accn. No.</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left">Eudicots</td>
<td align="left">Asterales</td>
<td align="left">
<italic>Artemisia dranunculus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402491">GQ402491</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Helianthus annuus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AM183222">AM183222</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Zinnia violacea</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AU304033">AU304033</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Brassicales</td>
<td align="left">
<italic>Arabidopsis thaliana</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="Y08501">Y08501</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Brassica napus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AP006444">AP006444</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Carica papaya</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="EU431224">EU431224</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Caryophyllales</td>
<td align="left">
<italic>Beta vulgaris</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="BA000009">BA000009</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Ericales</td>
<td align="left">
<italic>Actinidia deliciosa</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="FG448607">FG448607</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Pouteria sapota</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402492">GQ402492</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Sarracenia purpurea</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402493">GQ402493</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Gentianales</td>
<td align="left">
<italic>Ceropegia woodii</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402494">GQ402494</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Coffea arabica</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402495">GQ402495</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Pentas lanceolata</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402496">GQ402496</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Geraniales</td>
<td align="left">
<italic>Geranium sanguineum</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402497">GQ402497</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Lamiales</td>
<td align="left">
<italic>Digitalis purpurea</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402498">GQ402498</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Mimulus guttatus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402499">GQ402499</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Malpighiales</td>
<td align="left">
<italic>Breynia disticha</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402500">GQ402500</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Euphorbia tirucalli</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402501">GQ402501</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Populus trichocarpa</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="XM_002332800">XM_002332800</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Malvales</td>
<td align="left">
<italic>Theobroma cacao</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="CU593370">CU593370</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Myrtales</td>
<td align="left">
<italic>Eucalyptus gunnii</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="CT984759">CT984759</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Ranunculales</td>
<td align="left">
<italic>Papaver somniferum</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="FG608946">FG608946</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Rosales</td>
<td align="left">
<italic>Prunus dulcis</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="BU574137">BU574137</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Sapindales</td>
<td align="left">
<italic>Citrus sinensis</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402502">GQ402502</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Solanales</td>
<td align="left">
<italic>Ipomoea sp.</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402503">GQ402503</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Nicotiana tabacum</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="BA000042">BA000042</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Petunia axillaris</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="FN003412">FN003412</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Solanum lycopersicum</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="FJ374974">FJ374974</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Vitales</td>
<td align="left">
<italic>Cissus tuberosa</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402504">GQ402504</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Vitis vinifera</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="FM179380">FM179380</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">Monocots</td>
<td align="left">Asparagales</td>
<td align="left">
<italic>Agave americana</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402511">GQ402511</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Poales</td>
<td align="left">
<italic>Bambusa oldhamii</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="EU365401">EU365401</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Oryza sativa</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="BA000029">BA000029</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Sorghum bicolor</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="DQ984518">DQ984518</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Tripsacum dactyloides</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="DQ984517">DQ984517</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Triticum aestivum</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AP008982">AP008982</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Zea mays</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AY506529">AY506529</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Zingiberales</td>
<td align="left">
<italic>Maranta leuconeura</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402513">GQ402513</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Strelitzia reginae</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402512">GQ402512</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">Magnoliids</td>
<td align="left">Laurales</td>
<td align="left">
<italic>Persea sp.</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402505">GQ402505</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Piperales</td>
<td align="left">
<italic>Aristolochia elegans</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402506">GQ402506</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Peperomia sp.</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402507">GQ402507</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">Gymnosperms</td>
<td align="left">Coniferales</td>
<td align="left">
<italic>Picea glauca</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="EX322775">EX322775</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Podocarpus macrophyllus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402514">GQ402514</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Cycadales</td>
<td align="left">
<italic>Cycas taitungensis</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AP009381">AP009381</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Dioon edule</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402508">GQ402508</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td align="left">
<italic>Zamia pumila</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402509">GQ402509</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Ginkgoales</td>
<td align="left">
<italic>Ginkgo biloba</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="GQ402510">GQ402510</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Gnetales</td>
<td align="left">
<italic>Welwitschia mirabilis</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="DT601028">DT601028</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">Monilophytes</td>
<td align="left">Filicales</td>
<td align="left">
<italic>Adiantum capillus-veneris</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="DK949045">DK949045</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">Bryophytes</td>
<td align="left">Dendrocerotales</td>
<td align="left">
<italic>Megaceros aenigmaticus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="EU660574">EU660574</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Funariales</td>
<td align="left">
<italic>Physcomitrella patens</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AB251495">AB251495</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Marchantiales</td>
<td align="left">
<italic>Marchantia polymorpha</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="M68929">M68929</ext-link>
</td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td align="left">Charophytes</td>
<td align="left">Charales</td>
<td align="left">
<italic>Chara vulgaris</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AY267353">AY267353</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Chlorokybales</td>
<td align="left">
<italic>Chlorokybus atmophyticus</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="EF463011">EF463011</ext-link>
</td>
</tr>
<tr>
<td></td>
<td align="left">Coleochaetales</td>
<td align="left">
<italic>Chaetosphaeridium globosum</italic>
</td>
<td align="left">
<ext-link ext-link-type="gen" xlink:href="AF494279">AF494279</ext-link>
</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>Sequences were aligned using Muscle 3.7 [
<xref ref-type="bibr" rid="B50">50</xref>
] and manually adjusted in BioEdit 7.0.9 [
<xref ref-type="bibr" rid="B51">51</xref>
]. Edit sites were identified by comparison of DNA sequences with cDNA and/or EST sequences. To examine levels of functional constraint, poorly-aligned regions were first identified and removed using Gblocks 0.91b [
<xref ref-type="bibr" rid="B52">52</xref>
], then pairwise
<italic>d</italic>
<sub>N </sub>
and
<italic>d</italic>
<sub>S </sub>
were computed in MEGA 4.0.2 [
<xref ref-type="bibr" rid="B53">53</xref>
] using the Nei-Gojobori Model with a Jukes-Cantor correction for multiple hits.</p>
</sec>
<sec>
<title>Note added in proof</title>
<p>Another group has independently discovered the
<italic>rpl10 </italic>
gene in the mitochondrial genome of plants [
<xref ref-type="bibr" rid="B54">54</xref>
]. Similar to our study, Kubo and Arimura find that the mitochondrial gene is widely distributed among plants, is transcribed and RNA edited in multiple species, and has been lost from several lineages, including
<italic>Arabidopsis </italic>
and rice. These authors suggest, as we do, that a duplicated copy of the nucleus-encoded chloroplast
<italic>rpl10 </italic>
gene has functionally replaced the lost mitochondrial
<italic>rpl10 </italic>
gene independently in
<italic>Arabidopsis </italic>
and rice. For both species, they experimentally show that these putative mitochondrially-functioning L10 proteins have targeting signals that indeed induce localization to the mitochondrion, and also the chloroplast.</p>
</sec>
<sec>
<title>Authors' contributions</title>
<p>JPM conceived of the study, performed experimental and computational work, analyzed results, prepared figures and tables, and drafted the manuscript. LB also performed computational analyses, analyzed results, and helped draft the manuscript. Both authors read and approved the final manuscript.</p>
</sec>
</body>
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<sec>
<title>Acknowledgements</title>
<p>We thank Dana Ahmed, Sidra Jawaid, and Derek Schmidt for assistance with DNA and RNA isolations. This work was supported by start-up funds from the University of Nebraska Lincoln (JPM) and by the Natural Sciences and Engineering Research Council of Canada (LB).</p>
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