nMAT4, a maturase factor required for nad1 pre‐mRNA processing and maturation,is essential for holocomplex I biogenesis in Arabidopsis mitochondria

Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self‐splicing ribozyme and its own intron‐encoded maturase protein. A hallmark of maturases is that they are intron‐specific, acting as cofactors that bind their intron‐containing pre‐RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron‐encoded maturase open reading frames suggest that their splicing in vivo is assisted by ‘trans’‐acting protein factors. Interestingly, angiosperms harbor several nuclear‐encoded maturase‐related (nMat) genes that contain N‐terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.     

nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.     

Genome-wide identification and analysis of membrane-bound O-acyltransferase (MBOAT) gene family in plants

Membrane bound O-acyl transferase (MBOAT) family is composed of gene members encoding a variety of acyltransferase enzymes, which play important roles in plant acyl lipid metabolism. Here, we present the first genome-enabled identification and analysis of MBOAT gene models in plants. In total, we identified 136 plant MBOAT sequences from 14 plant species with complete genomes. Phylogenetic relationship analyses suggested the plant MBOAT gene models fell into four major groups, two of which likely encode enzymes of diacylglycerol acyltransferase 1 (DGAT1) and lysophospholipid acyltransferase (LPLAT), respectively, with one–three copies of paralogs present in each of the most plant species. A group of gene sequences, which are homologous to Saccharomyces cerevisiae glycerol uptake proteins (GUP), was identified in plants; copy numbers were conserved, with only one copy represented in each of the most plant species; analyses showed that residues essential for acyltransferases were more prone to be conserved than vertebrate orthologs. Among four groups, one was inferred to emerge in land plants and experience a rapid expansion in genomes of angiosperms, which suggested their important roles in adaptation of plants in lands. Sequence and phylogeny analyses indicated that genes in all four groups encode enzymes with acyltransferases. Comprehensive sequence identification of MBOAT family members and investigation into classification provide a complete picture of the MBOAT gene family in plants, and could shed light into enzymatic functions of different MBOAT genes in plants.     

Synonymous Codon Usage Bias in Plant Mitochondrial Genes Is Associated with Intron Number and Mirrors Species Evolution

Synonymous codon usage bias (SCUB) is a common event that a non-uniform usage of codons often occurs in nearly all organisms. We previously found that SCUB is correlated with both intron number and exon position in the plant nuclear genome but not in the plastid genome; SCUB in both nuclear and plastid genome can mirror the evolutionary specialization. However, how about the rules in the mitochondrial genome has not been addressed. Here, we present an analysis of SCUB in the mitochondrial genome, based on 24 plant species ranging from algae to land plants. The frequencies of NNA and NNT (A- and T-ending codons) are higher than those of NNG and NNC, with the strongest preference in bryophytes and the weakest in land plants, suggesting an association between SCUB and plant evolution. The preference for NNA and NNT is more evident in genes harboring a greater number of introns in land plants, but the bias to NNA and NNT exhibits even among exons. The pattern of SCUB in the mitochondrial genome differs in some respects to that present in both the nuclear and plastid genomes.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

Identification of shared single copy nuclear genes in Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various taxonomic levels

Background

Although the overwhelming majority of genes found in angiosperms are members of gene families, and both gene- and genome-duplication are pervasive forces in plant genomes, some genes are sufficiently distinct from all other genes in a genome that they can be operationally defined as 'single copy'. Using the gene clustering algorithm MCL-tribe, we have identified a set of 959 single copy genes that are shared single copy genes in the genomes of Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa. To characterize these genes, we have performed a number of analyses examining GO annotations, coding sequence length, number of exons, number of domains, presence in distant lineages, such as Selaginella and Physcomitrella, and phylogenetic analysis to estimate copy number in other seed plants and to demonstrate their phylogenetic utility. We then provide examples of how these genes may be used in phylogenetic analyses to reconstruct organismal history, both by using extant coverage in EST databases for seed plants and de novo amplification via RT-PCR in the family Brassicaceae.

Results

There are 959 single copy nuclear genes shared in Arabidopsis, Populus, Vitis and Oryza ["APVO SSC genes"]. The majority of these genes are also present in the Selaginella and Physcomitrella genomes. Public EST sets for 197 species suggest that most of these genes are present across a diverse collection of seed plants, and appear to exist as single or very low copy genes, though exceptions are seen in recently polyploid taxa and in lineages where there is significant evidence for a shared large-scale duplication event. Genes encoding proteins localized in organelles are more commonly single copy than expected by chance, but the evolutionary forces responsible for this bias are unknown. Regardless of the evolutionary mechanisms responsible for the large number of shared single copy genes in diverse flowering plant lineages, these genes are valuable for phylogenetic and comparative analyses. Eighteen of the APVO SSC single copy genes were amplified in the Brassicaceae using RT-PCR and directly sequenced. Alignments of these sequences provide improved resolution of Brassicaceae phylogeny compared to recent studies using plastid and ITS sequences. An analysis of sequences from 13 APVO SSC genes from 69 species of seed plants, derived mainly from public EST databases, yielded a phylogeny that was largely congruent with prior hypotheses based on multiple plastid sequences. Whereas single gene phylogenies that rely on EST sequences have limited bootstrap support as the result of limited sequence information, concatenated alignments result in phylogenetic trees with strong bootstrap support for already established relationships. Overall, these single copy nuclear genes are promising markers for phylogenetics, and contain a greater proportion of phylogenetically-informative sites than commonly used protein-coding sequences from the plastid or mitochondrial genomes.

Conclusions

Putatively orthologous, shared single copy nuclear genes provide a vast source of new evidence for plant phylogenetics, genome mapping, and other applications, as well as a substantial class of genes for which functional characterization is needed. Preliminary evidence indicates that many of the shared single copy nuclear genes identified in this study may be well suited as markers for addressing phylogenetic hypotheses at a variety of taxonomic levels.     

Evolution of Red Algal Plastid Genomes: Ancient Architectures,Introns, Horizontal Gene Transfer,and Taxonomic Utility of Plastid Markers

Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase – a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.     

Discovery of the rpl10 Gene in Diverse Plant Mitochondrial Genomes and Its Probable Replacement by the Nuclear Gene for Chloroplast RPL10 in Two Lineages of Angiosperms

Mitochondrial genomes of plants are much larger than those of mammals and often contain conserved open reading frames (ORFs) of unknown function. Here, we show that one of these conserved ORFs is actually the gene for ribosomal protein L10 (rpl10) in plant. No rpl10 gene has heretofore been reported in any mitochondrial genome other than the exceptionally gene-rich genome of the protist Reclinomonas americana. Conserved ORFs corresponding to rpl10 are present in a wide diversity of land plant and green algal mitochondrial genomes. The mitochondrial rpl10 genes are transcribed in all nine land plants examined, with five seed plant genes subject to RNA editing. In addition, mitochondrial-rpl10-like cDNAs were identified in EST libraries from numerous land plants. In three lineages of angiosperms, rpl10 is either lost from the mitochondrial genome or a pseudogene. In two of them (Brassicaceae and monocots), no nuclear copy of mitochondrial rpl10 is identifiably present, and instead a second copy of nuclear-encoded chloroplast rpl10 is present. Transient assays using green fluorescent protein indicate that this duplicate gene is dual targeted to mitochondria and chloroplasts. We infer that mitochondrial rpl10 has been functionally replaced by duplicated chloroplast counterparts in Brassicaceae and monocots.     

Dating the early evolution of plants: detection and molecular clock analyses of orthologs

Orthologs generally are under selective pressure against loss of function, while paralogs usually accumulate mutations and finally die or deviate in terms of function or regulation. Most ortholog detection methods contaminate the resulting datasets with a substantial amount of paralogs. Therefore we aimed to implement a straightforward method that allows the detection of ortholog clusters with a reduced amount of paralogs from completely sequenced genomes. The described cross-species expansion of the reciprocal best BLAST hit method is a time-effective method for ortholog detection, which results in 68% truly orthologous clusters and the procedure specifically enriches single-copy orthologs. The detection of true orthologs can provide a phylogenetic toolkit to better understand evolutionary processes. In a study across six photosynthetic eukaryotes, nuclear genes of putative mitochondrial origin were shown to be over-represented among single copy orthologs. These orthologs are involved in fundamental biological processes like amino acid metabolism or translation. Molecular clock analyses based on this dataset yielded divergence time estimates for the red/green algae (1,142 MYA), green algae/land plant (725 MYA), mosses/seed plant (496 MYA), gymno-/angiosperm (385 MYA) and monocotyledons/core eudicotyledons (301 MYA) divergence times. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prospects on the evolutionary mitogenomics of plants: A case study on the olive family (Oleaceae)

The mitogenome is rarely used to reconstruct the evolutionary history of plants, contrary to nuclear and plastid markers. Here, we evaluate the usefulness of mitochondrial DNA for molecular evolutionary studies in Oleaceae, in which cases of cytoplasmic male sterility (CMS) and of potentially contrasted organelle inheritance are known. We compare the diversity and the evolution of mitochondrial and chloroplast genomes by focusing on the olive complex and related genera. Using high‐throughput techniques, we reconstructed complete mitogenomes (ca. 0.7 Mb) and plastomes (ca. 156 kb) for six olive accessions and one Chionanthus. A highly variable organization of mitogenomes was observed at the species level. In olive, two specific chimeric genes were identified in the mitogenome of lineage E3 and may be involved in CMS. Plastid‐derived regions (mtpt) were observed in all reconstructed mitogenomes. Through phylogenetic reconstruction, we demonstrate that multiple integrations of mtpt regions have occurred in Oleaceae, but mtpt regions shared by all members of the olive complex derive from a common ancestor. We then assembled 52 conserved mitochondrial gene regions and complete plastomes of ten additional accessions belonging to tribes Oleeae, Fontanesieae and Forsythieae. Phylogenetic congruence between topologies based on mitochondrial regions and plastomes suggests a strong disequilibrium linkage between both organellar genomes. Finally, while phylogenetic reconstruction based on plastomes fails to resolve the evolutionary history of maternal olive lineages in the Mediterranean area, their phylogenetic relationships were successfully resolved with complete mitogenomes. Overall, our study demonstrates the great potential of using mitochondrial DNA in plant phylogeographic and metagenomic studies.     

Frequent mitochondrial gene introgression among high elevation Tibetan megophryid frogs revealed by conflicting gene genealogies

Historical mitochondrial introgression causes differences between a species' mitochondrial gene genealogy and its nuclear gene genealogy, making tree-based species delineation ambiguous. Using sequence data from one mitochondrial gene (cytochrome b ) and three nuclear genes (introns), we examined the evolutionary history of four high elevation Tibetan megophryid frog species, Scutiger boulengeri , Scutiger glandulatus , Scutiger mammatus and Scutiger tuberculatus . The three nuclear genes shared a similar history but the mitochondrial gene tree suggested a drastically different evolutionary scenario. The conflicts between them were explained by multiple episodes of mitochondrial introgression events via historical interspecific hybridization. 'Foreign' mitochondrial genomes might have been fixed in populations and extended through a large portion of the species' distribution. Some hybridization events were probably as old as 10 Myr, while others were recent. An F₁ hybrid was also identified. Historical hybridization events among the four species appeared to be persistent and were not restricted to the period of Pleistocene glaciation, as in several other well-studied cases. Furthermore, hybridization involved several species and occurred in multiple directions, and there was no indication of one mitochondrial genome being superior to others. In addition, incomplete lineage sorting resulting from budding speciation may have also explained some discrepancies between the mitochondrial DNA and nuclear gene trees. Combining all evidences, the former ' Scutiger mammatus ' appeared to be two species, including a new species. With the availability of a wide range of highly variable nuclear gene markers, we recommend using a combination of mitochondrial gene and multiple nuclear genes to reveal a complete species history.     

Parallel Loss of Plastid Introns and Their Maturase in the Genus Cuscuta

Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.     

Trans-splicing group II introns in plant mitochondria: the complete set of cis-arranged homologs in ferns, fern allies, and a hornwort.

The fragmentation of group II introns without concomitant loss of splicing competence is illustrated by extraordinary gene arrangements in plant mitochondrial genomes. The mitochondrial genes nad1, nad2, and nad5, all encoding subunits of the NADH dehydrogenase, require trans-splicing for functional assembly of their mRNAs in flowering plants. Tracing the origins of trans-splicing group II introns shows that they have evolved from formerly cis-arranged homologs whose descendants can still be identified in lineages of early branching land plants. In this contribution we present the full set of ancestor introns for all five conserved mitochondrial trans-splicing positions. These introns are strikingly small in the quillwort Isoetes lacustris, the continuous nad2 gene intron in this species representing the smallest (389 nt) land plant group II intron yet identified. cDNA analysis shows correct splicing of the introns in vivo and also identifies frequent RNA editing events in the flanking nad gene exons. Other representatives of the ancestral cis-arranged introns are identified in the fern Osmunda regalis, the horsetail Equisetum telmateia, and the hornwort Anthoceros crispulus. Only the now identified intron in Osmunda carries significant traces of a former maturase reading frame. The identification of a continuous homolog in Anthoceros demonstrates that intron invasion into the affected genes in some cases predated the split of vascular and nonvascular plants more than 400 million years ago. As an alternative to disruption after size increase, the respective introns can get secondarily lost in certain lineages.     

Fast evolution of the retroprocessed mitochondrial rps3 gene in Conifer II and further evidence for the phylogeny of gymnosperms

The popular view that plant mitochondrial genome evolves slowly in sequence has been recently challenged by the extraordinarily high substitution rates of mtDNA documented mainly from several angiosperm genera, but high substitution rate acceleration accompanied with great length variation has been very rarely reported in plant mitochondrial genes. Here, we studied evolution of the mitochondrial rps3 gene that encodes the ribosomal small subunit protein 3 and found a dramatically high variation in both length and sequence of an exon region of it in Conifer II. A sequence comparison between cDNA and genomic DNA showed that there are no RNA editing sites in the Conifer II rps3 gene. Southern blotting analyses of the total DNA and mtDNA, together with the real-time PCR analysis, showed that rps3 exists as a single mitochondrial locus in gymnosperms. It is very likely that the Conifer II rps3 gene has experienced retroprocessing, i.e., the re-integration of its cDNA into the mitochondrial genome, followed by an evolutionary acceleration due to the intron loss. In addition, the phylogenetic analysis of rps3 supports the sister relationship between conifers and Gnetales. In particular, the monophyly of conifer II is strongly supported by the shared loss of two rps3 introns. Our results also indicate that the mitochondrial gene tree would be affected in topology when the “edited” paralogs are analyzed together with their genomic sequences.