The molecular basis for allelic differences suggests Restorer-of-fertility 1 is a complex locus in sugar beet (Beta vulgaris L.).

BACKGROUND: Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production in many crops. Sugar beet CMS is associated with a unique mitochondrial protein named preSATP6 that forms a 250-kDa complex. Restorer-of-fertility 1 (Rf1) is a nuclear gene that suppresses CMS and is, hence, one of the targets of sugar beet breeding. Rf1 has dominant, semi-dominant and recessive alleles, suggesting that it may be a multi-allelic locus; however, the molecular basis for differences in genetic action is obscure. Molecular cloning of Rf1 revealed a gene (orf20) whose protein products produced in transgenics can bind with preSATP6 to generate a novel 200-kDa complex. The complex is also detected in fertility-restored anthers concomitant with a decrease in the amount of the 250-kDa complex. Molecular diversity of the Rf1 locus involves organizational diversity of a gene cluster composed of orf20-like genes (RF-Oma1s). We examined the possibility that members of the clustered RF-Oma1 in this locus could be associated with fertility restoration. RESULTS: Six yet uncharacterized RF-Oma1s from dominant and recessive alleles were examined to determine whether they could generate the 200-kDa complex. Analyses of transgenic calli revealed that three RF-Oma1s from a dominant allele could generate the 200-kDa complex, suggesting that clustered RF-Oma1s in the dominant allele can participate in fertility restoration. None of the three copies from two recessive alleles was 200-kDa generative. The absence of this ability was confirmed by analyzing mitochondrial complexes in anthers of plants having these recessive alleles. Together with our previous data, we designed a set of PCR primers specific to the 200-kDa generative RF-Oma1s. The amount of mRNA measured by this primer set inversely correlated with the amount of the 250-kDa complex in anthers and positively correlated with the strength of the Rf1 alleles. CONCLUSIONS: Fertility restoration by sugar beet Rf1 can involve multiple RF-Oma1s clustered in the locus, implying that stacking 200-kDa generative copies in the locus strengthens the efficacy, whereas the absence of 200-kDa generative copies in the locus makes the allele recessive irrespective of the copy number. We propose that sugar beet Rf1 is a complex locus.

A Lineage-Specific Paralog of Oma1 Evolved into a Gene Family from Which a Suppressor of Male Sterility-Inducing Mitochondria Emerged in Plants.

Cytoplasmic male sterility (MS) in plants is caused by MS-inducing mitochondria, which have emerged frequently during plant evolution. Nuclear restorer-of-fertility (Rf)genes can suppress their cognate MS-inducing mitochondria. Whereas many Rfs encode a class of RNA-binding protein, the sugar beet (Caryophyllales) Rf encodes a protein resembling Oma1, which is involved in the quality control of mitochondria. In this study, we investigated the molecular evolution of Oma1 homologs in plants. We analyzed 37 plant genomes and concluded that a single copy is the ancestral state in Caryophyllales. Among the sugar beet Oma1 homologs, the orthologous copy is located in a syntenic region that is preserved in Arabidopsis thaliana. The sugar beet Rf is a complex locus consisting of a small Oma1 homolog family (RF-Oma1 family) unique to sugar beet. The gene arrangement in the vicinity of the locus is seen in some but not all Caryophyllalean plants and is absent from Ar. thaliana. This suggests a segmental duplication rather than a whole-genome duplication as the mechanism of RF-Oma1 evolution. Of thirty-seven positively selected codons in RF-Oma1, twenty-six of these sites are located in predicted transmembrane helices. Phylogenetic network analysis indicated that homologous recombination among the RF-Oma1 members played an important role to generate protein activity related to suppression. Together, our data illustrate how an evolutionarily young Rf has emerged from a lineage-specific paralog. Interestingly, several evolutionary features are shared with the RNA-binding protein type Rfs. Hence, the evolution of the sugar beet Rf is representative of Rf evolution in general.

Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris).

KEY MESSAGE: The sugar beet Rf1 locus has a number of molecular variants. We found that one of the molecular variants is a weak allele of a previously identified allele. Male sterility (MS) caused by nuclear-mitochondrial interaction is called cytoplasmic male sterility (CMS) in which MS-inducing mitochondria are suppressed by a nuclear gene, restorer-of-fertility. Rf and rf are the suppressing and non-suppressing alleles, respectively. This dichotomic view, however, seems somewhat unsatisfactory to explain the recently discovered molecular diversity of Rf loci. In the present study, we first identified sugar beet line NK-305 as a new source of Rf1. Our crossing experiment revealed that NK-305 Rf1 is likely a semi-dominant allele that restores partial fertility when heterozygous but full fertility when homozygous, whereas Rf1 from another sugar beet line appeared to be a dominant allele. Proper degeneration of anther tapetum is a prerequisite for pollen development; thus, we compared tapetal degeneration in the NK-305 Rf1 heterozygote and the homozygote. Degeneration occurred in both genotypes but to a lesser extent in the heterozygote, suggesting an association between NK-305 Rf1 dose and incompleteness of tapetal degeneration leading to partial fertility. Our protein analyses revealed a quantitative correlation between NK-305 Rf1 dose and a reduction in the accumulation of a 250 kDa mitochondrial protein complex consisting of a CMS-specific mitochondrial protein encoded by MS-inducing mitochondria. The abundance of Rf1 transcripts correlated with NK-305 Rf1 dose. The molecular organization of NK-305 Rf1 suggested that this allele evolved through intergenic recombination. We propose that the sugar beet Rf1 locus has a series of multiple alleles that differ in their ability to restore fertility and are reflective of the complexity of Rf evolution.

A fertility-restoring genotype of beet (Beta vulgaris L.) is composed of a weak restorer-of-fertility gene and a modifier gene tightly linked to the Rf1 locus.

Cytoplasmic male sterility (CMS) is a plant trait that involves interactions between nuclear- and mitochondrial genomes. In CMS, the nuclear restorer-of-fertility gene (Rf), a suppressor of male-sterility inducing mitochondria, is one of the best known genetic factors. Other unidentified genetic factors may exist but have not been well characterized. In sugar beet (Beta vulgaris L.), CMS is used for hybrid seed production, but few male-sterility inducing nuclear genotypes exist. Such genotypes could be introduced from a closely related plant such as leaf beet, but first the fertility restoring genotype of the related plant must be characterized. Here, we report the discovery of a Japanese leaf beet accession 'Fukkoku-ouba' that has both male-sterility inducing and fertility restoring genotypes. We crossed the leaf beet accession with a sugar beet CMS line, developed succeeding generations, and examined the segregation of two DNA markers that are linked to two sugar beet Rfs, Rf1 and Rf2. Only the Rf2 marker co-segregated with fertility restoration in every generation, implying that the Rf1 locus in leaf beet is occupied by a non-restoring allele. Fertility restoration was incomplete without a genetic factor closely linked to Rf1, leading to the assumption that the Rf1 locus encodes a modifier that cannot restore fertility by itself but perhaps strengthens another Rf. We sequenced the apparently non-restoring 'Fukkoku-ouba' rf1 gene-coding region and found that it closely resembles a restoring allele. The protein product demonstrated its potential to suppress CMS in transgenic suspension cells. In contrast, 'Fukkoku-ouba' rf1 transcript abundance was highly reduced compared to that of the restoring Rf1. Consistently, changes in protein complexes containing CMS-associated mitochondrial protein in anthers were very minor. Accordingly, we concluded that 'Fukkoku-ouba' rf1 is a hypomorph that acts as a non-restoring allele but has the potential to support another Rf, i.e. it is a modifier candidate.

Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.).

BACKGROUND: Obtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line 'NK-219mm-O' is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis. FINDINGS: Five sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross, the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat. CONCLUSIONS: Efficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.

Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris).

Genetic conflict between cytoplasmically inherited elements and nuclear genes arising from their different transmission patterns can be seen in cytoplasmic male sterility (CMS), the mitochondrion-encoded inability to shed functional pollen. CMS is associated with a mitochondrial open reading frame (ORF) that is absent from non-sterility inducing mitochondria (S-orf). Nuclear genes that suppress CMS are called restorer-of-fertility (Rf) genes. Post-transcriptional and translational repression of S-orf mediates the molecular action of Rf that encodes a class of RNA-binding proteins with pentatricopeptide repeat (PPR) motifs. Besides the PPR-type of Rfs, there are also non-PPR Rfs, but the molecular interactions between non-PPR Rf and S-orf have not been described. In this study, we investigated the interaction of bvORF20, a non-PPR Rf from sugar beet (Beta vulgaris), with preSatp6, the S-orf from sugar beet. Anthers expressing bvORF20 contained a protein that interacted with preSATP6 protein. Analysis of anthers and transgenic calli expressing a FLAG-tagged bvORF20 suggested the binding of preSATP6 to bvORF20. To see the effect of bvORF20 on preSATP6, which exists as a 250-kDa protein complex in CMS plants, signal bands of preSATP6 in bvORF20-expressing and non-expressing anthers were compared by immunoblotting combined with Blue Native polyacrylamide gel electrophoresis. The signal intensity of the 250-kDa band decreased significantly, and 200- and 150-kDa bands appeared in bvORF20-expressing anthers. Transgenic callus expressing bvORF20 also generated the 200- and 150-kDa bands. The 200-kDa complex is likely to include both preSATP6 and bvORF20. Post-translational interaction between preSATP6 and bvORF20 appears to alter the higher order structure of preSATP6 that may lead to fertility restoration in sugar beet.