An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae

The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced that it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a handful of potential mitosomal proteins were found to be encoded in the complete genome of the microsporidian Encephalitozoon cuniculi, and significantly no proteins of the mitochondrial carrier family were identified. These carriers facilitate the transport of solutes across the inner mitochondrial membrane, are a means of communication between the mitochondrion and cytosol, and are abundant in organisms with aerobic mitochondria. Here, we report the characterization of a mitochondrial carrier protein in the microsporidian Antonospora locustae and demonstrate that the protein is heterologously targeted to mitochondria in Saccharomyces cerevisiae. The protein is phylogenetically allied to the NAD⁺ transporter of S. cerevisiae, but we show that it has high specificity for ATP and ADP when expressed in Escherichia coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.     

The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and Evolution of Mitochondria

Mitochondrial DNA (mtDNA) is unquestionably the remnant of an α-proteobacterial genome, yet only ∼10%–20% of mitochondrial proteins are demonstrably α-proteobacterial in origin (the “α-proteobacterial component,” or APC). The evolutionary ancestry of the non-α-proteobacterial component (NPC) is obscure and not adequately accounted for in current models of mitochondrial origin. I propose that in the host cell that accommodated an α-proteobacterial endosymbiont, much of the NPC was already present, in the form of a membrane-bound metabolic organelle (the premitochondrion) that compartmentalized many of the non-energy-generating functions of the contemporary mitochondrion. I suggest that this organelle also possessed a protein import system and various ion and small-molecule transporters. In such a scenario, an α-proteobacterial endosymbiont could have been converted relatively directly and rapidly into an energy-generating organelle that incorporated the extant metabolic functions of the premitochondrion. This model (the “pre-endosymbiont hypothesis”) effectively represents a synthesis of previous, contending mitochondrial origin hypotheses, with the bulk of the mitochondrial proteome (much of the NPC) having an endogenous origin and the minority component (the APC) having a xenogenous origin.Considering the central role played in all eukaryotic cells by mitochondria or mitochondrion-related organelles (MROs, such as hydrogenosomes and mitosomes) (Hjort et al. 2010; Shiflett and Johnson 2010; Müller et al. 2012), the question of the origin and subsequent evolution of the mitochondrion has long captivated and challenged biologists. In a recent article in this series (Gray 2012), I discussed in detail several aspects of mitochondrial evolution, focusing particularly on how well the accumulating molecular data can be accommodated in current models of mitochondrial origin. In this context, the origin and evolution of the mitochondrial proteome, as opposed to the origin and evolution of the mitochondrial genome, were examined from the perspective of comparative mitochondrial proteomics. Somewhat disconcertingly, as more data have become available, we find ourselves considerably less certain about key aspects of how mitochondria originated than we were (or thought we were) several decades ago.Here, I summarize key points discussed in more detail in the previous article before presenting a novel perspective on how the mitochondrion might have originated. The new model proposed here, which represents a synthesis of both endogenous (“origin from within”) and xenogenous (“origin from outside”) modes, is advanced in an attempt to account for the inability of a purely endosymbiotic model, whose strongest support has come from studies of the mitochondrial genome, to adequately accommodate data on the mitochondrial proteome.     

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus)

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.     

Genome-Wide Analysis of the Phosphoinositide Kinome from Two Ciliates Reveals Novel Evolutionary Links for Phosphoinositide Kinases in Eukaryotic Cells

Background

The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs) that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms.

Methodology/Principal Findings

We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates.

Conclusions/Significance

Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP₃ signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs) not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory pathways of phosphoinositides may be more widespread than previously thought in unicellular organisms.     

The incredible shrinking organelle

The mitochondrion is probably the evolutionary remnant of a bacterial symbiont, yet contemporary mitochondria are nothing like contemporary bacteria. Evolutionary shrinkage of the mitochondrial genome is well documented, but what about wholesale shrinkage of the organelle itself?Considering its central role in energy metabolism in almost all eukaryotes, the mitochondrion is an amazingly plastic organelle, both evolutionarily and functionally. The few genes that the mitochondrial genome (mitochondrial DNA; mtDNA) encodes are clearly bacterial in origin—emanating from the α-proteobacterial lineage—supporting the widely held view that the mitochondrion is the evolutionary remnant of a bacterial symbiont (Gray et al, 2001). However, contemporary mitochondria are nothing like contemporary bacteria. For one thing, even the most gene-rich mtDNA encodes far less genetic information than the most gene-poor bacterial genome, and mitochondrial genomes are different from bacterial genomes in form, organization and mode of expression; these features vary tremendously among diverse eukaryotes. Mitochondrial genomes might be circular, linear or even highly fragmented, and they might contain highly fragmented and rearranged genes. Only within a poorly studied group of eukaryotic microbes—protists—known as jakobid flagellates does the mtDNA resemble a typical, albeit highly reduced, bacterial genome.In addition, the mitochondrial proteome is not only overwhelmingly (>90%) encoded in the nucleus, but only a small proportion (10–15%) is demonstrably α-proteobacterial in evolutionary affiliation. Thus, in the evolutionary transition from bacterial symbiont to integrated organelle, the mitochondrion has undergone an impressive degree of re-tailoring, shedding the bulk of its genetic information and taking on proteins of diverse evolutionary origins. Moreover, this re-tailoring is highly variable within different eukaryotic lineages, with an intriguing chunk of the mitochondrial proteome seeming to be organism-specific—lacking demonstrable sequence homologues other than in very close evolutionary relatives.Although the evolutionary shrinkage of the mitochondrial genome is well-documented, what is less widely appreciated is the wholesale shrinkage of the organelle itself in certain anaerobic eukaryotes. Taken to its extreme, such shrinkage involves complete loss of the mitochondrial genome, with a consequent reduction in the structural complexity and biochemical versatility of the organelle. This simplification might include elimination of the electron-transport chain (ETC) and thus lead to inability of the resulting mitochondrion-related organelle (MRO) to carry out a key function of aerobic mitochondria: ATP synthesis through coupled oxidative phosphorylation (for a full account, see Hjort et al, 2010).One such MRO, the hydrogenosome, is a hydrogen-producing organelle that was originally characterized in an anaerobic protist, Trichomonas vaginalis. The T. vaginalis hydrogenosome lacks mtDNA as well as components of the classic mitochondrial ETC, relying instead on substrate-level phosphorylation to generate ATP. Initially, the resemblance between the anaerobic biochemistry of the T. vaginalis MRO and that of anaerobic bacteria such as Clostridia raised the possibility that the hydrogenosome might have a different evolutionary origin than the classic aerobic mitochondrion. However, studies of hydrogenosomal proteins have demonstrated that the hydrogenosome is an evolutionarily derived (remnant) mitochondrion. Hydrogenosomes have been found in eukaryotes that are widely separated in phylogenetic trees, and in such trees, anaerobic, hydrogenosome-containing eukaryotes are often interspersed with close relatives that grow aerobically and contain conventional mitochondria. This punctate phylogenetic distribution suggests that the transition from mitochondrion to hydrogenosome has happened repeatedly and independently throughout eukaryotic evolution.The mitosome, an even more shrunken MRO that has not only dispensed entirely with a genome, but also has no ATP-generating capacity. This MRO was discovered in anaerobic eukaryotes that were initially thought to lack mitochondria entirely, the postulate being that they diverged away from the main line of eukaryotic evolution prior to the symbiosis that led to the mitochondrion. However, in all supposedly amitochondriate protists that have been examined, a candidate mitosome has been identified. As with hydrogenosomes, a punctate phylogenetic distribution of mitosomes is emerging.Recently, intermediate forms of ''shrinking organelle'' have been identified in the anaerobic protists Nyctotherus ovalis, Blastocystis sp. and Proteromonas lacertae (Hjort et al, 2010; Pérez-Brocal et al, 2010; de Graaf et al, 2011), relatives of brown algae and diatoms. In these cases, regions of the mtDNA that code for terminal portions of the ETC and for the mitochondrial ATP synthase have been discarded. The remaining DNA specifies genes for components of a mitochondrial translation system, as well as subunits of a proton-pumping complex I (NADH:ubiquinone oxidoreductase); a remarkable example—comparing the ciliate Nyctotherus with the stramenopiles Blastocystsis or Proteromonas—of convergent mtDNA evolution. These observations suggest that the transitional MROs of Nyctotherus, Blastocystis and Proteromonas retain a partial ETC, as well as the ability to synthesize protein, whereas other data (EST surveys) indicate that they are metabolically more complex than either hydrogenosomes or mitosomes. The discovery of these particular MROs is important because their existence argues that the transition from fully fledged aerobic mitochondrion to fully fledged anaerobic mitosome proceeds through, and might stop at, several intermediate stages: a realization that not only dramatically emphasizes the evolutionary and functional versatility of the mitochondrion, but also opens the possibility that we might yet uncover still other variations of this incredible shrinking organelle.     

Complete sequence of the mitochondrial genome of Tetrahymena thermophila and comparative methods for identifying highly divergent genes

The complete sequence of the mitochondrial genome of Tetrahymena thermophila has been determined and compared with the mitochondrial genome of Tetrahymena pyriformis. The sequence similarity clearly indicates homology of the entire T.thermophila and T.pyriformis mitochondrial genomes. The T.thermophila genome is very compact, most of the intergenic regions are short (only three are longer than 63 bp) and comprise only 3.8% of the genome. The nad9 gene is tandemly duplicated in T.thermophila. Long terminal inverted repeats and the nad9 genes are undergoing concerted evolution. There are 55 putative genes: three ribosomal RNA genes, eight transfer RNA genes, 22 proteins with putatively assigned functions and 22 additional open reading frames of unknown function. In order to extend indications of homology beyond amino acid sequence similarity we have examined a number of physico-chemical properties of the mitochondrial proteins, including theoretical pI, molecular weight and particularly the predicted transmembrane spanning regions. This approach has allowed us to identify homologs to ymf58 (nad4L), ymf62 (nad6) and ymf60 (rpl6).     

Diversity and reductive evolution of mitochondria among microbial eukaryotes

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.     

The origin of mitochondria in light of a fluid prokaryotic chromosome model

Biologists agree that the ancestor of mitochondria was an α-proteobacterium. But there is no consensus as to what constitutes an α-proteobacterial gene. Is it a gene found in all or several α-proteobacteria, or in only one? Here, we examine the proportion of α-proteobacterial genes in α-proteobacterial genomes by means of sequence comparisons. We find that each α-proteobacterium harbours a particular collection of genes and that, depending upon the lineage examined, between 97 and 33% are α-proteobacterial by the nearest-neighbour criterion. Our findings bear upon attempts to reconstruct the mitochondrial ancestor and upon inferences concerning the collection of genes that the mitochondrial ancestor possessed at the time that it became an endosymbiont.     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)     

The completed macronuclear genome of a model ciliate Tetrahymena thermophila and its application in genome scrambling and copy number analyses

The ciliate Tetrahymena thermophila has been a powerful model system for molecular and cellular biology. However, some investigations have been limited due to the incomplete closure and sequencing of the macronuclear genome assembly, which for many years has been stalled at 1,158 scaffolds, with large sections of unknown sequences(available in Tetrahymena Genome Database, TGD, http://ciliate.org/). Here we completed the first chromosome-level Tetrahymena macronuclear genome assembly, with approximately 300× long Single Molecule, Real-Time reads of the wild-type SB210 cells—the reference strain for the initial macronuclear genome sequencing project. All 181 chromosomes were capped with two telomeres and gaps were entirely closed. The completed genome shows significant improvements over the current assembly(TGD 2014) in both chromosome structure and sequence integrity. The majority of previously identified gene models shown in TGD were retained,with the addition of 36 new genes and 883 genes with modified gene models. The new genome and annotation were incorporated into TGD. This new genome allows for pursuit in some underexplored areas that were far more challenging previously; two of them, genome scrambling and chromosomal copy number, were investigated in this study. We expect that the completed macronuclear genome will facilitate many studies in Tetrahymena biology, as well as multiple lines of research in other eukaryotes.     

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity

The FtsH proteases, also called AAA proteases, are membrane-bound ATP-dependent metalloproteases. The Arabidopsis genome contains a total of 12 FtsH-like genes. Two of them, AtFtsH4 and AtFtsH11, encode proteins with a high similarity to Yme1p, a subunit of the i-AAA complex in yeast mitochondria. Phylogenetic analysis groups the AtFtsH4, AtFtsH11 and Yme1 proteins together, with AtFtsH4 being the most similar to Yme1. Using immunological method we demonstrate here that AtFtsH4 is an exclusively mitochondrial protein while AtFtsH11 is found in both chloroplasts and mitochondria. AtFtsH4 and AtFtsH11 proteases are integral parts of the inner mitochondrial membrane and expose their catalytic sites towards the intermembrane space, same as yeast i-AAA. Database searches revealed that orthologs of AtFtsH4 and AtFtsH11 are present in both monocotyledonous and dicotyledonous plants. The two plant i-AAA proteases differ significantly in their termini: the FtsH4 proteins have a characteristic alanine stretch at the C-terminal end while FtsH11s have long N-terminal extensions. Blue-native gel electrophoresis revealed that AtFtsH4 and AtFtsH11 form at least two complexes with apparent molecular masses of about 1500 kDa. This finding implies that plants, in contrast to fungi and metazoa, have more than one complex with a topology similar to that of yeast i-AAA.     

Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.     

Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena

A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.     

Origin and Evolution of the Mitochondrial Proteome

The endosymbiotic theory for the origin of mitochondria requires substantial modification. The three identifiable ancestral sources to the proteome of mitochondria are proteins descended from the ancestral α-proteobacteria symbiont, proteins with no homology to bacterial orthologs, and diverse proteins with bacterial affinities not derived from α-proteobacteria. Random mutations in the form of deletions large and small seem to have eliminated nonessential genes from the endosymbiont-mitochondrial genome lineages. This process, together with the transfer of genes from the endosymbiont-mitochondrial genome to nuclei, has led to a marked reduction in the size of mitochondrial genomes. All proteins of bacterial descent that are encoded by nuclear genes were probably transferred by the same mechanism, involving the disintegration of mitochondria or bacteria by the intracellular membranous vacuoles of cells to release nucleic acid fragments that transform the nuclear genome. This ongoing process has intermittently introduced bacterial genes to nuclear genomes. The genomes of the last common ancestor of all organisms, in particular of mitochondria, encoded cytochrome oxidase homologues. There are no phylogenetic indications either in the mitochondrial proteome or in the nuclear genomes that the initial or subsequent function of the ancestor to the mitochondria was anaerobic. In contrast, there are indications that relatively advanced eukaryotes adapted to anaerobiosis by dismantling their mitochondria and refitting them as hydrogenosomes. Accordingly, a continuous history of aerobic respiration seems to have been the fate of most mitochondrial lineages. The initial phases of this history may have involved aerobic respiration by the symbiont functioning as a scavenger of toxic oxygen. The transition to mitochondria capable of active ATP export to the host cell seems to have required recruitment of eukaryotic ATP transport proteins from the nucleus. The identity of the ancestral host of the α-proteobacterial endosymbiont is unclear, but there is no indication that it was an autotroph. There are no indications of a specific α-proteobacterial origin to genes for glycolysis. In the absence of data to the contrary, it is assumed that the ancestral host cell was a heterotroph.     

Use of Tetrahymena thermophila To Study the Role of Protozoa in Inactivation of Viruses in Water

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4°C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.     

Suppression of Repeat-Mediated Gross Mitochondrial Genome Rearrangements by RecA in the Moss Physcomitrella patens

RecA and its ubiquitous homologs are crucial components in homologous recombination. Besides their eukaryotic nuclear counterparts, plants characteristically possess several bacterial-type RecA proteins localized to chloroplasts and/or mitochondria, but their roles are poorly understood. Here, we analyzed the role of the only mitochondrial RecA in the moss Physcomitrella patens. Disruption of the P. patens mitochondrial recA gene RECA1 caused serious defects in plant growth and development and abnormal mitochondrial morphology. Analyses of mitochondrial DNA in disruptants revealed that frequent DNA rearrangements occurred at multiple loci. Structural analysis suggests that the rearrangements, which in some cases were associated with partial deletions and amplifications of mitochondrial DNA, were due to aberrant recombination between short (<100 bp) direct and inverted repeats in which the sequences were not always identical. Such repeats are abundant in the mitochondrial genome, and interestingly many are located in group II introns. These results suggest that RECA1 does not promote but rather suppresses recombination among short repeats scattered throughout the mitochondrial genome, thereby maintaining mitochondrial genome stability. We propose that RecA-mediated homologous recombination plays a crucial role in suppression of short repeat-mediated genome rearrangements in plant mitochondria.