Genes for two mitochondrial ribosomal proteins in flowering plants are derived from their chloroplast or cytosolic counterparts

Often during flowering plant evolution, ribosomal protein genes have been lost from the mitochondrion and transferred to the nucleus. Here, we show that substitution by a duplicated, divergent gene originally encoding the chloroplast or cytosolic ribosomal protein counterpart accounts for two missing mitochondrial genes in diverse angiosperms. The rps13 gene is missing from the mitochondrial genome of many rosids, and a transferred copy of this gene is not evident in the nucleus of Arabidopsis, soybean, or cotton. Instead, these rosids contain a divergent nuclear copy of an rps13 gene of chloroplast origin. The product of this gene from all three rosids was shown to be imported into isolated mitochondria but not into chloroplasts. The rps8 gene is missing from the mitochondrion and nucleus of all angiosperms examined. A divergent copy of the gene encoding its cytosolic counterpart (rps15A) was identified in the nucleus of four angiosperms and one gymnosperm. The product of this gene from Arabidopsis and tomato was imported successfully into mitochondria. We infer that rps13 was lost from the mitochondrial genome and substituted by a duplicated nuclear gene of chloroplast origin early in rosid evolution, whereas rps8 loss and substitution by a gene of nuclear/cytosolic origin occurred much earlier, in a common ancestor of angiosperms and gymnosperms.     

Evolution of mitochondrial gene content: gene loss and transfer to the nucleus

Mitochondrial gene content is highly variable across extant eukaryotes. The number of mitochondrial protein genes varies from 3 to 67, while tRNA gene content varies from 0 to 27. Moreover, these numbers exclude the many diverse lineages of non-respiring eukaryotes that lack a mitochondrial genome yet still contain a mitochondrion, albeit one often highly derived in ultrastructure and metabolic function, such as the hydrogenosome. Diversity in tRNA gene content primarily reflects differential usage of imported tRNAs of nuclear origin. In the case of protein genes, most of this diversity reflects differential degrees of functional gene transfer to the nucleus, with more minor contributions resulting from gene loss from the cell as a consequence of either substitution via a functional nuclear homolog or the cell's dispensation of the function of the gene product. The tempo and pattern of mitochondrial gene loss is highly episodic, both across the broad sweep of eukaryotes and within such well-studied groups as angiosperms. All animals, some plants, and certain other groups of eukaryotes are mired in profound stases in mitochondrial gene content, whereas other lineages have experienced relatively frequent gene loss. Loss and transfer to the nucleus of ribosomal protein and succinate dehydrogenase genes has been especially frequent, sporadic, and episodic during angiosperm evolution. Potential mechanisms for activation of transferred genes have been inferred, and intermediate stages in the process have been identified by comparative studies. Several hypotheses have been proposed for why mitochondrial genes are transferred to the nucleus, why mitochondria retain genomes, and why functional gene transfer is almost exclusively unidirectional.     

Evolutionary transfers of mitochondrial genes to the nucleus in the Populus lineage and coexpression of nuclear and mitochondrial Sdh4 genes

The transfer of mitochondrial genes to the nucleus is an ongoing evolutionary process in flowering plants. Evolutionarily recent gene transfers provide insights into the evolutionary dynamics of the process and the way in which transferred genes become functional in the nucleus. Genes that are present in the mitochondrion of some angiosperms but have been transferred to the nucleus in the Populus lineage were identified by searches of Populus sequence databases. Sequence analyses and expression experiments were used to characterize the transferred genes. Two succinate dehydrogenase genes and six mitochondrial ribosomal protein genes have been transferred to the nucleus in the Populus lineage and have become expressed. Three transferred genes have gained an N-terminal mitochondrial targeting presequence from other pre-existing genes and two of the transferred genes do not contain an N-terminal targeting presequence. Intact copies of the succinate dehydrogenase gene Sdh4 are present in both the mitochondrion and the nucleus. Both copies of Sdh4 are expressed in multiple organs of two Populus species and RNA editing occurs in the mitochondrial copy. These results provide a genome-wide perspective on mitochondrial genes that were transferred to the nucleus and became expressed, functional genes during the evolutionary history of Populus.     

Multiple losses and transfers to the nucleus of two mitochondrial succinate dehydrogenase genes during angiosperm evolution.

Unlike in animals, the functional transfer of mitochondrial genes to the nucleus is an ongoing process in plants. All but one of the previously reported transfers in angiosperms involve ribosomal protein genes. Here we report frequent transfer of two respiratory genes, sdh3 and sdh4 (encoding subunits 3 and 4 of succinate dehydrogenase), and we also show that these genes are present and expressed in the mitochondria of diverse angiosperms. Southern hybridization surveys reveal that sdh3 and sdh4 have been lost from the mitochondrion about 40 and 19 times, respectively, among the 280 angiosperm genera examined. Transferred, functional copies of sdh3 and sdh4 were characterized from the nucleus in four and three angiosperm families, respectively. The mitochondrial targeting presequences of two sdh3 genes are derived from preexisting genes for anciently transferred mitochondrial proteins. On the basis of the unique presequences of the nuclear genes and the recent mitochondrial gene losses, we infer that each of the seven nuclear sdh3 and sdh4 genes was derived from a separate transfer to the nucleus. These results strengthen the hypothesis that angiosperms are experiencing a recent evolutionary surge of mitochondrial gene transfer to the nucleus and reveal that this surge includes certain respiratory genes in addition to ribosomal protein genes.     

Expression of floral MADS-box genes in basal angiosperms: implications for the evolution of floral regulators

The ABC model of floral organ identity is based on studies of Arabidopsis and Antirrhinum, both of which are highly derived eudicots. Most of the genes required for the ABC functions in Arabidopsis and Antirrhinum are members of the MADS-box gene family, and their orthologs are present in all major angiosperm lineages. Although the eudicots comprise 75% of all angiosperms, most of the diversity in arrangement and number of floral parts is actually found among basal angiosperm lineages, for which little is known about the genes that control floral development. To investigate the conservation and divergence of expression patterns of floral MADS-box genes in basal angiosperms relative to eudicot model systems, we isolated several floral MADS-box genes and examined their expression patterns in representative species, including Amborella (Amborellaceae), Nuphar (Nymphaeaceae) and Illicium (Austrobaileyales), the successive sister groups to all other extant angiosperms, plus Magnolia and Asimina, members of the large magnoliid clade. Our results from multiple methods (relative-quantitative RT-PCR, real-time PCR and RNA in situ hybridization) revealed that expression patterns of floral MADS-box genes in basal angiosperms are broader than those of their counterparts in eudicots and monocots. In particular, (i) AP1 homologs are generally expressed in all floral organs and leaves, (ii) AP3/PI homologs are generally expressed in all floral organs and (iii) AG homologs are expressed in stamens and carpels of most basal angiosperms, in agreement with the expectations of the ABC model; however, an AG homolog is also expressed in the tepals of Illicium. The broader range of strong expression of AP3/PI homologs is inferred to be the ancestral pattern for all angiosperms and is also consistent with the gradual morphological intergradations often observed between adjacent floral organs in basal angiosperms.     

Mitochondrial sequence migrated downstream to a nuclear V-ATPase B gene is transcribed but non-functional.

A promiscuous nuclear sequence containing a mitochondrial DNA fragment was isolated from rice. Nucleotide sequence analysis reveals that the cDNA clone #21 carries a mitochondrial sequence homologous to the 3' portion of the rps19 gene followed by the 5' portion of the rps3 gene. The mitochondrial sequence is present in an antisense orientation. Sequence comparison of the #21 cDNA with the original mitochondrial sequence shows 99% similarity, suggesting a recent transfer event. Moreover, evidence for a lack of an RNA editing event and retaining of the group II intron sequence strongly suggests that the sequence was transferred from mitochondrion to the nucleus via DNA rather than RNA as an intermediate. The upstream region to the mitochondria-derived sequence shows homology to part of the vacuolar H(+)-ATPase B subunit (V-ATPase B) gene. Isolation of a functional V-ATPase B cDNA and its comparison with the #21 cDNA reveal a number of nucleotide substitutions resulting in many translational stop codons in the #21 cDNA. This indicates that the #21 cDNA sequence is not functional. Analysis of genomic sequences shows the presence of five intron sequences in the #21 cDNA, whereas the functional V-ATPase B gene has 14 introns. Of these, three exons and their internal two introns are homologous to each other, suggesting a duplication event of V-ATPase B genomic DNA. The results of this investigation strongly suggest that the mitochondrial sequence was integrated in an antisense orientation into the pre-existing V-ATPase B pseudogene that can be transcribed and spliced. This represents a case of unsuccessful gene transfer from mitochondrion to the nucleus.     

Adaptations required for mitochondrial import following mitochondrial to nucleus gene transfer of ribosomal protein S10

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphipathic alpha-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal.     

A rice <Emphasis Type="Italic">YABBY</Emphasis> gene, <Emphasis Type="Italic">OsYABBY4</Emphasis>, preferentially expresses in developing vascular tissue

Developmental gene families have diversified during land plant evolution. The primary role of YABBY gene family is promoting abaxial fate in model eudicot, Arabidopsis thaliana. However recent results suggest that roles of YABBY genes are not conserved in the angiosperms. In this paper, a rice YABBY gene was isolated, and its expression patterns were analyzed in detail. Sequence characterization and phylogenetic analyses showed the gene is OsYABBY4, which is group-classified into FIL/YAB3 subfamily. Beta-glucuronidase reporter assay and in situ analysis consistently revealed that OsYABBY4 was expressed in the meristems and developing vascular tissue of rice, predominantly in the phloem tissue, suggesting that the function of the rice gene is different from those of its counterparts in eudicots. OsYABBY4 may have been recruited to regulate the development of vasculature in rice. However, transgenic Arabidopsis plants ectopically expressing OsYABBY4 behaved very like those over-expressing FIL or YAB3 with abaxialized lateral organs, suggesting the OsYABBY4 protein domain is conserved with its Arabidopsis counterparts in sequences. Our results also indicate that the functional diversification of OsYABBY4 may be associated with the divergent spatial-temporal expression patterns, and YABBY family members may have preserved different expression regulatory systems and functions during the evolution of different kinds of species.     

Recruitment of CRABS CLAW to promote nectary development within the eudicot clade

Nectaries are secretory organs that are widely present in flowering plants that function to attract floral pollinators. Owing to diversity in nectary positions and structures, they are thought to have originated multiple times during angiosperm evolution, with their potential contribution to the diversification of flowering plants and pollinating animals being considerable. We investigated the genetic basis of diverse nectary forms in eudicot angiosperm species using CRABS CLAW (CRC), a gene required for nectaries in Arabidopsis. CRC expression is conserved in morphologically different nectaries from several core eudicot species and is required for nectary development in both rosids and asterids, two major phylogenetic lineages of eudicots. However, in a basal eudicot species, no evidence of CRC expression in nectaries was found. Considering the phylogenetic distribution of nectary positions and CRC expression analyses in eudicots, we propose that diverse nectaries in core eudicots share conserved CRC gene regulation, and that derived nectary positions in eudicots have altered regulation of CRC. As the ancestral function of CRC lies in the regulation of carpel development, it may have been co-opted as a regulator of nectary development within the eudicots, concomitant with the association of nectaries with reproductive organs in derived lineages.     

Isolation and characterization of the C-class MADS-box gene from the distylous pseudo-cereal <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

In the model species Arabidopsis thaliana, the floral homeotic C-class gene AGAMOUS (AG) specifies reproductive organ (stamen and carpels) identity and floral meristem determinacy. Gene function analyses in other core eudicots species reveal functional conservation, subfunctionalization and function switch of the C-lineage in this clade. To identify the possible roles of AG-like genes in regulating floral development in distylous species with dimorphic flowers (pin and thrum) and the C function evolution, we isolated and identified an AG ortholog from Fagopyrum esculentum (buckwheat, Family Polygonaceae), an early diverging species of core eudicots preceding the rosids-asterids split. Protein sequence alignment and phylogenetic analysis grouped FaesAG into the euAG lineage. Expression analysis suggested that FaesAG expressed exclusively in developing stamens and gynoecium of pin and thrum flowers. Moreover, FaesAG expression reached a high level in both pin and thrum flowers at the time when the stamens were undergoing rapidly increased in size and microspore mother cells were in meiosis. FaesAG was able to substitute for the endogenous AG gene in specifying stamen and carpel identity and in an Arabidopsis ag-1 mutant. Ectopic expression of FaesAG led to very early flowering, and produced a misshapen inflorescence and abnormal flowers in which sepals had converted into carpels and petals were converted to stamens. Our results confirmed establishment of the complete C-function of the AG orthologous gene preceding the rosids-asterids split, despite the distinct floral traits present in early- and late-diverging lineages of core eudicot angiosperms.     

Conservation of B-class floral homeotic gene function between maize and Arabidopsis

The ABC model of flower development, established through studies in eudicot model species, proposes that petal and stamen identity are under the control of B-class genes. Analysis of B- and C-class genes in the grass species rice and maize suggests that the C- and B-class functions are conserved between monocots and eudicots, with B-class genes controlling stamen and lodicule development. We have undertaken a further analysis of the maize B-class genes Silky1, the putative AP3 ortholog, and Zmm16, a putative PI ortholog, in order to compare their function with the Arabidopsis B-class genes. Our results show that maize B-class proteins interact in vitro to bind DNA as an obligate heterodimer, as do Arabidopsis B-class proteins. The maize proteins also interact with the appropriate Arabidopsis B-class partner proteins to bind DNA. Furthermore, we show that maize B-class genes are capable of rescuing the corresponding Arabidopsis B-class mutant phenotypes. This demonstrates B-class activity of the maize gene Zmm16, and provides compelling evidence that B-class gene function is conserved between monocots and eudicots.     

A horizontally transferred tRNA(Cys) gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is aminoacylated

Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.