APOK3, a pollen killer antidote in Arabidopsis thaliana.

The principles of heredity state that the two alleles carried by a heterozygote are equally transmitted to the progeny. However, genomic regions that escape this rule have been reported in many organisms. It is notably the case of genetic loci referred to as gamete killers, where one allele enhances its transmission by causing the death of the gametes that do not carry it. Gamete killers are of great interest, particularly to understand mechanisms of evolution and speciation. Although being common in plants, only a few, all in rice, have so far been deciphered to the causal genes. Here, we studied a pollen killer found in hybrids between two accessions of Arabidopsis thaliana. Exploring natural variation, we observed this pollen killer in many crosses within the species. Genetic analyses revealed that three genetically linked elements are necessary for pollen killer activity. Using mutants, we showed that this pollen killer works according to a poison-antidote model, where the poison kills pollen grains not producing the antidote. We identified the gene encoding the antidote, a chimeric protein addressed to mitochondria. De novo genomic sequencing in 12 natural variants with different behaviors regarding the pollen killer revealed a hyper variable locus, with important structural variations particularly in killer genotypes, where the antidote gene recently underwent duplications. Our results strongly suggest that the gene has newly evolved within A. thaliana. Finally, we identified in the protein sequence polymorphisms related to its antidote activity.

A restorer-of-fertility-like pentatricopeptide repeat protein promotes cytoplasmic male sterility in Arabidopsis thaliana.

Pentatricopeptide repeat (PPR) proteins form a large family of proteins targeted to organelles, where they post-transcriptionally modulate gene expression through binding to specific RNA sequences. Among them, the mitochondria-targeted restorer-of-fertility (Rf) PPRs inhibit peculiar mitochondrial genes that are detrimental to male gametes and cause cytoplasmic male sterility (CMS). Here, we revealed three nuclear loci involved in CMS in a cross between two distant Arabidopsis thaliana strains, Sha and Cvi-0. We identified the causal gene at one of these loci as RFL24, a conserved gene encoding a PPR protein related to known Rf PPRs. By analysing fertile revertants obtained in a male sterile background, we demonstrate that RFL24 promotes pollen abortion, in contrast with the previously described Rf PPRs, which allow pollen to survive in the presence of a sterilizing cytoplasm. We show that the sterility caused by the RFL24 Cvi-0 allele results from higher expression of the gene during early pollen development. Finally, we predict a binding site for RFL24 upstream of two mitochondrial genes, the CMS gene and the important gene cob. These results suggest that the conservation of RFL24 is linked to a primary role of ensuring a proper functioning of mitochondria, and that it was subsequently diverted by the CMS gene to its benefit.

The Consequences of a Disruption in Cyto-Nuclear Coadaptation on the Molecular Response to a Nitrate Starvation in Arabidopsis.

Mitochondria and chloroplasts are important actors in the plant nutritional efficiency. So, it could be expected that a disruption of the coadaptation between nuclear and organellar genomes impact plant response to nutrient stresses. We addressed this issue using two Arabidopsis accessions, namely Ct1 and Jea, and their reciprocal cytolines possessing the nuclear genome from one parent and the organellar genomes of the other one. We measured gene expression, and quantified proteins and metabolites under N starvation and non-limiting conditions. We observed a typical response to N starvation at the phenotype and molecular levels. The phenotypical response to N starvation was similar in the cytolines compared to the parents. However, we observed an effect of the disruption of genomic coadaptation at the molecular levels, distinct from the previously described responses to organellar stresses. Strikingly, genes differentially expressed in cytolines compared to parents were mainly repressed in the cytolines. These genes encoded more mitochondrial and nuclear proteins than randomly expected, while N starvation responsive ones were enriched in genes for chloroplast and nuclear proteins. In cytolines, the non-coadapted cytonuclear genomic combination tends to modulate the response to N starvation observed in the parental lines on various biological processes.

Novel Cytonuclear Combinations Modify Arabidopsis thaliana Seed Physiology and Vigor.

Dormancy and germination vigor are complex traits of primary importance for adaptation and agriculture. Intraspecific variation in cytoplasmic genomes and cytonuclear interactions were previously reported to affect germination in Arabidopsis using novel cytonuclear combinations that disrupt co-adaptation between natural variants of nuclear and cytoplasmic genomes. However, specific aspects of dormancy and germination vigor were not thoroughly explored, nor the parental contributions to the genetic effects. Here, we specifically assessed dormancy, germination performance and longevity of seeds from Arabidopsis plants with natural and new genomic compositions. All three traits were modified by cytonuclear reshuffling. Both depth and release rate of dormancy could be modified by a changing of cytoplasm. Significant changes on dormancy and germination performance due to specific cytonuclear interacting combinations mainly occurred in opposite directions, consistent with the idea that a single physiological consequence of the new genetic combination affected both traits oppositely. However, this was not always the case. Interestingly, the ability of parental accessions to contribute to significant cytonuclear interactions modifying the germination phenotype was different depending on whether they provided the nuclear or cytoplasmic genetic compartment. The observed deleterious effects of novel cytonuclear combinations (in comparison with the nuclear parent) were consistent with a contribution of cytonuclear interactions to germination adaptive phenotypes. More surprisingly, we also observed favorable effects of novel cytonuclear combinations, suggesting suboptimal genetic combinations exist in natural populations for these traits. Reduced sensitivity to exogenous ABA and faster endogenous ABA decay during germination were observed in a novel cytonuclear combination that also exhibited enhanced longevity and better germination performance, compared to its natural nuclear parent. Taken together, our results strongly support that cytoplasmic genomes represent an additional resource of natural variation for breeding seed vigor traits.

Genomic Conflicts that Cause Pollen Mortality and Raise Reproductive Barriers in Arabidopsis thaliana.

Species differentiation and the underlying genetics of reproductive isolation are central topics in evolutionary biology. Hybrid sterility is one kind of reproductive barrier that can lead to differentiation between species. Here, we analyze the complex genetic basis of the intraspecific hybrid male sterility that occurs in the offspring of two distant natural strains of Arabidopsis thaliana, Shahdara and Mr-0, with Shahdara as the female parent. Using both classical and quantitative genetic approaches as well as cytological observation of pollen viability, we demonstrate that this particular hybrid sterility results from two causes of pollen mortality. First, the Shahdara cytoplasm induces gametophytic cytoplasmic male sterility (CMS) controlled by several nuclear loci. Second, several segregation distorters leading to allele-specific pollen abortion (pollen killers) operate in hybrids with either cytoplasm. The complete sterility of the hybrid with the Shahdara cytoplasm results from the genetic linkage of the two causes of pollen mortality, i.e., CMS nuclear determinants and pollen killers. Furthermore, natural variation at these loci in A. thaliana is associated with different male-sterility phenotypes in intraspecific hybrids. Our results suggest that the genomic conflicts that underlie segregation distorters and CMS can concurrently lead to reproductive barriers between distant strains within a species. This study provides a new framework for identifying molecular mechanisms and the evolutionary history of loci that contribute to reproductive isolation, and possibly to speciation. It also suggests that two types of genomic conflicts, CMS and segregation distorters, may coevolve in natural populations.

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field.

Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics ofA. thaliana.

A cryptic cytoplasmic male sterility unveils a possible gynodioecious past for Arabidopsis thaliana.

Gynodioecy, the coexistence of hermaphrodites and females (i.e. male-sterile plants) in natural plant populations, most often results from polymorphism at genetic loci involved in a particular interaction between the nuclear and cytoplasmic genetic compartments (cytonuclear epistasis): cytoplasmic male sterility (CMS). Although CMS clearly contributes to the coevolution of involved nuclear loci and cytoplasmic genomes in gynodioecious species, the occurrence of CMS genetic factors in the absence of sexual polymorphism (cryptic CMS) is not easily detected and rarely taken in consideration. We found cryptic CMS in the model plant Arabidopsis thaliana after crossing distantly related accessions, Sha and Mr-0. Male sterility resulted from an interaction between the Sha cytoplasm and two Mr-0 genomic regions located on chromosome 1 and chromosome 3. Additional accessions with either nuclear sterility maintainers or sterilizing cytoplasms were identified from crosses with either Sha or Mr-0. By comparing two very closely related cytoplasms with different male-sterility inducing abilities, we identified a novel mitochondrial ORF, named orf117Sha, that is most likely the sterilizing factor of the Sha cytoplasm. The presence of orf117Sha was investigated in worldwide natural accessions. It was found mainly associated with a single chlorotype in accessions belonging to a clade predominantly originating from Central Asia. More than one-third of accessions from this clade carried orf117Sha, indicating that the sterilizing-inducing cytoplasm had spread in this lineage. We also report the coexistence of the sterilizing cytoplasm with a non-sterilizing cytoplasm at a small, local scale in a natural population; in addition a correlation between cytotype and nuclear haplotype was detected in this population. Our results suggest that this CMS system induced sexual polymorphism in A. thaliana populations, at the time when the species was mainly outcrossing.

The role of organelle genomes in plant adaptation: time to get to work!

That organellar genome variation can play a role in plant adaptation has been suggested by several lines of evidence, including cytoplasm capture, cytoplasm effects in local adaptation, and positive selection in a chloroplast gene. In-depth analysis and better understanding of the genetic basis of plant adaptation is becoming a main objective in plant science. Arabidopsis thaliana has all the required characteristics to be used as a model for obtaining knowledge on the mechanisms underlying the role of organelles in plant adaptation. The availability of the appropriate tools and materials for assessing organelle genetic variation will open up new opportunities for developing novel breeding strategies.

Cytoplasmic phylogeny and evidence of cyto-nuclear co-adaptation in Arabidopsis thaliana.

In recent years Arabidopsis thaliana has become a model species for genomic variability and adaptation studies. Although impressive quantities of data have been gathered on the nuclear genomic diversity of this species, little has been published regarding its cytoplasmic diversity. We analyzed the diversity of plastid (pt) and mitochondrial (mt) genomes among 95 accessions, covering most Arabidopsis geographic origins. Four intergenic regions of the pt genome were sequenced, and a total of 68 polymorphisms and 65 pt haplotypes were identified. Several strategies were developed to identify mt polymorphisms among a subset of 14 accessions. Fifteen polymorphisms were further developed as PCR-based markers and used to analyze the whole set of 95 accessions. Using statistical parsimony, we built pt and mt phylogenetic networks of haplotype groups. To root the pt network, the pt intergenic regions of two related Arabidopsis species, Arabidopsis lyrata and Arabidopsis arenosa, were also sequenced. The mt and pt phylogenies are highly congruent and could be combined into a single cytoplasmic phylogeny. To estimate whether co-adaptation between nuclear and cytoplasmic genomes exists in A. thaliana, we tested the germination capacity in challenging conditions of 27 pairs of reciprocal F(2) families. We found that the cytoplasm donor had a significant effect on the germination capacity of some F(2) families.

Sequence analysis of two alleles reveals that intra-and intergenic recombination played a role in the evolution of the radish fertility restorer (Rfo).

BACKGROUND: Land plant genomes contain multiple members of a eukaryote-specific gene family encoding proteins with pentatricopeptide repeat (PPR) motifs. Some PPR proteins were shown to participate in post-transcriptional events involved in organellar gene expression, and this type of function is now thought to be their main biological role. Among PPR genes, restorers of fertility (Rf) of cytoplasmic male sterility systems constitute a peculiar subgroup that is thought to evolve in response to the presence of mitochondrial sterility-inducing genes. Rf genes encoding PPR proteins are associated with very close relatives on complex loci. RESULTS: We sequenced a non-restoring allele (L7rfo) of the Rfo radish locus whose restoring allele (D81Rfo) was previously described, and compared the two alleles and their PPR genes. We identified a ca 13 kb long fragment, likely originating from another part of the radish genome, inserted into the L7rfo sequence. The L7rfo allele carries two genes (PPR-1 and PPR-2) closely related to the three previously described PPR genes of the restorer D81Rfo allele (PPR-A, PPR-B, and PPR-C). Our results indicate that alleles of the Rfo locus have experienced complex evolutionary events, including recombination and insertion of extra-locus sequences, since they diverged. Our analyses strongly suggest that present coding sequences of Rfo PPR genes result from intragenic recombination. We found that the 10 C-terminal PPR repeats in Rfo PPR gene encoded proteins result from the tandem duplication of a 5 PPR repeat block. CONCLUSIONS: The Rfo locus appears to experience more complex evolution than its flanking sequences. The Rfo locus and PPR genes therein are likely to evolve as a result of intergenic and intragenic recombination. It is therefore not possible to determine which genes on the two alleles are direct orthologs. Our observations recall some previously reported data on pathogen resistance complex loci.

The Ogura sterility-inducing protein forms a large complex without interfering with the oxidative phosphorylation components in rapeseed mitochondria.

The Ogura cytoplasmic male sterility causing protein, ORF138, was found to be part of a complex with an apparent size of over 750 kDa in the inner membrane of mitochondria of sterile plants. ORF138 did not colocalize with any of the oxidative phosphorylation complexes, nor did its presence modify their apparent size or amount, compared to samples from fertile isogenic plants. We attempted to detect potential proteins or nucleic acids that could be involved in the large ORF138 complex by 2D PAGE, immunoprecipitation and nuclease treatments of native extracts. All our results suggest that the ORF138 protein is the main, if not only, component of this large complex. The capacities of complexes I, II, IV, and ATP synthase were identical in samples from sterile and fertile plants. Isolated mitochondria from sterile plants showed a higher oxygen consumption than those from fertile plants. In vivo respiration measurements suggest that the difference in O(2) consumption measured at the organelle level is compensated at the cell/tissue level, completely in leaves, but only partially in male reproductive organs.

pur4 mutations are lethal to the male, but not the female, gametophyte and affect sporophyte development in Arabidopsis.

Purine metabolism is crucial in living cells and involves three complex pathways in plants: the de novo synthesis, the salvage, and the degradation pathways. The relative importance of each pathway in plant development and reproduction, however, is still unclear. We identified two T-DNA insertions in the Arabidopsis (Arabidopsis thaliana) PUR4 gene (At1g74260) that encodes formylglycinamidine ribonucleotide synthase (EC 6.3.5.3), the fourth enzyme in the de novo purine biosynthesis pathway. The mutated alleles were never transmitted through the pollen of heterozygous plants but could be inherited through the female gametophyte, indicating that de novo purine synthesis is specifically necessary for pollen development. Because the pur4 mutations were lethal to the male gametophyte, homozygous pur4 plants could not be obtained. However, the reproductive phenotype of hetererozygous plants carrying the pur4-2 mutated allele was more severe than that carrying the pur4-1 mutated allele, and pur4-2/+ plants showed slightly delayed early development. We showed that the pur4-2 allele produces an antisense transcript and that the amount of PUR4 mRNA is reduced in these plants. Transient expression of a translational fusion with the green fluorescent protein in Arabidopsis plantlets showed that the formylglycinamidine ribonucleotide synthase protein is dually targeted to chloroplast and mitochondria, suggesting that at least some steps of the de novo purine biosynthesis pathway can take place in both organelles in Arabidopsis, a dual location previously thought to be a peculiarity of ureide-forming tropical legumes.

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus).

Ogura cytoplasmic male sterility (CMS) occurs naturally in radish and has been introduced into rapeseed (Brassica napus) by protoplast fusion. As with all CMS systems, it involves a constitutively expressed mitochondrial gene which induces male sterility to otherwise hermaphroditic plants (so they become females) and a nuclear gene named restorer of fertility that restores pollen production in plants carrying a sterility-inducing cytoplasm. A correlative approach using light and electron microscopy was applied to define what stages throughout development were affected and the subcellular events leading to the abortion of the developing pollen grains upon the expression of the mitochondrial protein. Three central stages of development (tetrad, mid-microspore and vacuolate microspore) were compared between fertile, restored, and sterile plants. At each stage observed, the pollen in fertile and restored plants had similar cellular structures and organization. The deleterious effect of the sterility protein expression started as early as the tetrad stage. No typical mitochondria were identified in the tapetum at any developmental stage and in the vacuolate microspores of the sterile plants. In addition, some striking ultrastructural alterations of the cell's organization were also observed compared with the normal pattern of development. The results showed that Ogu-INRA CMS was due to premature cell death events of the tapetal cells, presumably by an autolysis process rather than a normal PCD, which impairs pollen development at the vacuolate microspore stage, in the absence of functional mitochondria.

Cytoplasmic suppression of Ogura cytoplasmic male sterility in European natural populations of Raphanus raphanistrum.

The Ogura cytoplasmic male sterility (CMS) of radish has been used for hybrid seed production in radish and Brassica crops. It is the only CMS system occurring in wild populations for which the gene responsible for sterility and a restorer gene have been formally identified. In Japan, gynodioecious populations of radish carrying Ogura or an Ogura-related cytoplasm have been described. The occurrence of restorer genes for the Ogura CMS in wild radish (Raphanus raphanistrum) in France led us to search for the corresponding male sterility gene (orf138) in several natural populations in France, England and Lebanon. We detected the orf138 gene, by PCR, at low frequency, in three populations from France and one from Southern England. Further molecular characterization showed that these plants carried a cytoplasm closely related to the original Ogura cytoplasm, with a variant orf138 coding sequence, previously reported to be ancestral. We performed crosses with sterile and maintainer radish lines, to test the ability of this wild Ogura-related cytoplasm to induce sterility. Surprisingly, the European Ogura-related cytoplasm did not cause sterility. Northern blots and circular RT-PCR analyses showed that orf138 gene expression was impaired in these plants because of a novel cytoplasm-dependent transcript-processing site.

The molecular biology of cytoplasmically inherited male sterility and prospects for its engineering.

Nucleocytoplasmic male sterilities are binary genetic systems driven by mitochondrial, maternally inherited genes that induce male sterility and a female phenotype and which are overcome by nuclear restorers of fertility. They contribute to the reproductive biology and evolution of natural populations and are valuable tools for the commercial production of hybrid seeds in crops. For species with no natural form of cytoplasmic male sterility, such sterility can in some cases be introduced from different, but related, species through sexual crosses or somatic hybridisation. Somatic hybridisation is the only technique currently available for manipulating plant mitochondrial genomes. Recent successes in plastid transformation have opened up entirely new perspectives for the engineering of cytoplasmic male sterilities in transplastomic plants.

Biochemical and functional characterization of ORF138, a mitochondrial protein responsible for Ogura cytoplasmic male sterility in Brassiceae.

In cytoplasmic male sterility (CMS), original mitochondrial genes contribute to sex determinism by provoking pollen abortion. The function of the encoded proteins remains unclear. We studied the ORF138 protein, responsible for the 'Ogura' CMS, which is both used in hybrid seed production and present in natural populations. We analyzed the biochemical and structural properties of this protein in male-sterile plants and in E. coli. We showed that this protein spontaneously forms dimers in vitro. Truncated variants of the protein, containing either the hydrophobic or the hydrophilic moiety, also spontaneously dimerize. By fractionating mitochondria, we showed that ORF138 was strongly associated with the inner mitochondrial membrane of male-sterile plants. Our results also strongly suggest that ORF138 forms oligomers in male-sterile plant mitochondria. In E. coli, ORF138 was associated with the plasma membrane, as shown by membrane fractionation, and formed oligomers. The production of this protein strongly inhibited bacterial growth, but not by inhibiting respiration. The observed toxic effects required both the hydrophilic and hydrophobic moieties of the protein.

Unveiling the molecular arms race between two conflicting genomes in cytoplasmic male sterility?

Cytoplasmic male sterility can be thought of as the product of a genetic conflict between two genomes that have different modes of inheritance. Male sterilizing factors, generally encoded by chimeric mitochondrial genes, can be down-regulated by specific nuclear restorer genes. The recent cloning of a restorer gene in rice and its comparison with restorer genes cloned in petunia and radish could be regarded as the beginning of a general molecular scenario in this peculiar arms race.

Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed, using the Arabidopsis genome sequence and radish genetic mapping.

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.

Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family.

Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level. The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.

The nucleo-mitochondrial conflict in cytoplasmic male sterilities revisited.

Cytoplasmic male sterility (CMS) in plants is a classical example of genomic conflict, opposing maternally-inherited cytoplasmic genes (mitochondrial genes in most cases), which induce male sterility, and nuclear genes, which restore male fertility. In natural populations, this type of sex control leads to gynodioecy, that is, the co-occurrence of female and hermaphroditic individuals within a population. According to theoretical models, two conditions may maintain male sterility in a natural population: (1) female advantage (female plants are reproductively more successful than hermaphrodites on account of their global seed production); (2) the counter-selection of nuclear fertility restorers when the corresponding male-sterility-inducing cytoplasm is lacking. In this review, we re-examine the model of nuclear-mitochondrial conflict in the light of recent experimental results from naturally occurring CMS, alloplasmic CMS (appearing after interspecific crosses resulting from the association of nuclear and cytoplasmic genomes from different species), and CMS plants obtained in the laboratory and carrying mitochondrial mutations. We raise new hypotheses and discuss experimental models that would take physiological interactions between cytoplasmic and nuclear genomes into account.