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.     

Distribution of introns in the mitochondrial gene nad1 in land plants: phylogenetic and molecular evolutionary implications

Forty-six species of diverse land plants were investigated by sequencing for their intron content in the mitochondrial gene nad1. A total of seven introns, all belonging to group II, were found, and two were newly discovered in this study. All 13 liverworts examined contain no intron, the same condition as in green algae. Mosses and hornworts, however, share one intron by themselves and another one with vascular plants. These intron distribution patterns are consistent with the hypothesis that liverworts represent the basal-most land plants and that the two introns were gained in the common ancestor of mosses-hornworts-vascular plants after liverworts had diverged. Hornworts also possess a unique intron of their own. A fourth intron was found only in Equisetum L., Marattiaceae, Ophioglossum L., Osmunda L., Asplenium L., and Adiantum L., and was likely acquired in their common ancestor, which supports the monophyly of moniliformopses. Three introns that were previously characterized in angiosperms and a few pteridophytes are now all extended to lycopods, and were likely gained in the common ancestor of vascular plants. Phylogenetic analyses of the intron sequences recovered topologies mirroring those of the plants, suggesting that the introns have all been vertically inherited. All seven nad1 group II introns show broad phylogenetic distribution patterns, with the narrowest being in moniliformopses and hornworts, lineages that date back to at least the Devonian (345 million years ago) and Silurian (435 million years ago), respectively. Hence, these introns must have invaded the genes via ancient transpositional events during the early stage of land plant evolution. Potentially heavy RNA editing was observed in nad1 of Haplomitrium Dedecek, Takakia Hatt. & Inoue, hornworts, Isoetes L., Ophioglossum, and Asplenium. A new nomenclature is proposed for group II introns.     

Exclusive conservation of mitochondrial group II intron nad4i548 among liverworts and its use for phylogenetic studies in this ancient plant clade

Liverworts occupy a pivotal position in land plant (embryophyte) phylogeny as the presumed earliest-branching major clade, sister to all other land plants, including the mosses, hornworts, lycophytes, monilophytes and seed plants. Molecular support for this earliest dichotomy in land plant phylogeny comes from strikingly different occurrences of introns in mitochondrial genes distinguishing liverworts from all other embryophytes. Exceptionally, however, the nad5 gene--the mitochondrial locus hitherto used most widely to elucidate early land plant phylogeny--carries a group I type intron that is shared between liverworts and mosses. We here explored whether a group II intron, the other major type of organellar intron, would similarly be conserved in position across the entire diversity of extant liverworts and could be of use for phylogenetic analyses in this supposedly most ancient embryophyte clade. To this end, we investigated the nad4 gene as a candidate locus possibly featuring different introns in liverworts as opposed to the non-liverwort embryophyte (NLE) lineage. We indeed found group II intron nad4i548 universally conserved in a wide phylogenetic sampling of 55 liverwort taxa, confirming clade specificity and surprising evolutionary stability of plant mitochondrial introns. As expected, intron nad4i548g2 carries phylogenetic information in its variable sequences, which confirms and extends previous cladistic insights on liverwort evolution. We integrate the new nad4 data with those of the previously established mitochondrial nad5 and the chloroplast rbcL and rps4 genes and present a phylogeny based on the fused datasets. Notably, the phylogenetic analyses suggest a reconsideration of previous phylogenetic and taxonomic assignments for the genera Calycularia and Mylia and resolve a sister group relationship of Ptilidiales and Porellales.     

Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses,liverworts, and hornworts) and the lycophytes

The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.     

Mosses share mitochondrial group II introns with flowering plants, not with liverworts

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.     

Variation in sequence and RNA editing within core domains of mitochondrial group II introns among plants

The 3' regions of several group II introns within the mitochondrial genes nad1 and nad7 show unexpected sequence divergence among flowering plants, and the core domains 5 and 6 are predicted to have weaker helical structure than those in self-splicing group II introns. To assess whether RNA editing improves helical stability by the conversion of A-C mispairs to A-U pairs, we sequenced RT-PCR amplification products derived from excised intron RNAs or partially spliced precursors. Only in some cases was editing observed to strengthen the predicted helices. Moreover, the editing status within nad1 intron 1 and nad7 intron 4 was seen to differ among plant species, so that homologous intron sequences shared lower similarity at the RNA level than at the DNA level. Plant-specific variation was also seen in the length of the linker joining domains 5 and 6 of nad7 intron 3; it ranged from 4 nt in wheat to 11 nt in soybean, in contrast to the 2-4 nt length typical of classical group II introns. However, this intron is excised as a lariat structure with a domain 6 branchpoint adenosine. Our observations suggest that the core structures and sequences of these plant mitochondrial introns are subject to less stringent evolutionary constraints than conventional group II introns.     

The complete mitochondrial DNA sequence of Mesostigma viride identifies this green alga as the earliest green plant divergence and predicts a highly compact mitochondrial genome in the ancestor of all green plants.

To gain insights into the nature of the mitochondrial genome in the common ancestor of all green plants, we have completely sequenced the mitochondrial DNA (mtDNA) of Mesostigma viride. This green alga belongs to a morphologically heterogeneous class (Prasinophyceae) that includes descendants of the earliest diverging green plants. Recent phylogenetic analyses of ribosomal RNAs (rRNAs) and concatenated proteins encoded by the chloroplast genome identified Mesostigma as a basal branch relative to the Streptophyta and the Chlorophyta, the two phyla that were previously thought to contain all extant green plants. The circular mitochondrial genome of Mesostigma resembles the mtDNAs of green algae occupying a basal position within the Chlorophyta in displaying a small size (42,424 bp) and a high gene density (86.6% coding sequences). It contains 65 genes that are conserved in other mtDNAs. Although none of these genes represents a novel coding sequence among green plant mtDNAs, four of them (rps1, sdh3, sdh4, and trnL[caa]) have not been reported previously in chlorophyte mtDNAs, and two others (rpl14 and trnI[gau]) have not been identified in the streptophyte mtDNAs examined so far (land-plant mtDNAs). Phylogenetic analyses of 19 concatenated mtDNA-encoded proteins favor the hypothesis that Mesostigma represents the earliest branch of green plant evolution. Four group I introns (two in rnl and two in cox1) and three group II introns (two in nad3 and one in cox2), two of which are trans-spliced at the RNA level, reside in Mesostigma mtDNA. The insertion sites of the three group II introns are unique to this mtDNA, suggesting that trans-splicing arose independently in the Mesostigma lineage and in the Streptophyta. The few structural features that can be regarded as ancestral in Mesostigma mtDNA predict that the common ancestor of all green plants had a compact mtDNA containing a minimum of 75 genes and perhaps two group I introns. Considering that the mitochondrial genome is much larger in size in land plants than in Mesostigma, we infer that mtDNA size began to increase dramatically in the Streptophyta either during the evolution of charophyte green algae or during the transition from charophytes to land plants.     

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.     

Cis- and trans-splicing of group II introns in plant mitochondria

Group II-type introns in the mitochondrial genes of flowering plants belong to the ribozymic, mobile retroelement family, but not all exhibit conventional structural features and some follow unusual splicing pathways. Moreover, several introns have been disrupted by DNA rearrangements, so that separately-transcribed precursors undergo splicing in trans. RNA processing in plant mitochondria has the added complexity of C-to-U RNA editing which also sometimes occurs within core intron structures or at exon sites very close to introns. It appears that mitochondrial introns in flowering plants have followed quite different evolutionary pathways than other group II introns.     

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.     

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphatedependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize(Zea mays) DEAD-box RNA helicase48(Zm RH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis,and seed development. Loss of Z...     

Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.