Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Twenty-nine mitochondrial genomes from 19 angiosperm species have been completely sequenced and have been found to vary in genome size and gene content. Seven of these mitochondrial genomes are known to induce cytoplasmic male sterility (CMS), and thus can be utilized for hybrid seed production or the prevention of pollen dispersal. Genome rearrangement frequently is observed in MS-inducing mitochondria, but it also occurs as part of the normal inter- or intraspecific variation in male fertile (MF) mitochondria. Sequence analyses have revealed that the repertoire of genuine genes is indistinguishable between MS-inducing and MF mitochondria. Deleterious mutations appear to be rare in MS-inducing mitochondria, which may be consistent with the lack of systemic manifestation of CMS. On the other hand, several nucleotide substitutions remain to be investigated for their potential mild effects. Various mitochondrial ORFs are associated with CMS (CMS-ORFs). There are some common but not strict features shared by CMS-ORFs such as their uniqueness to the CMS mitochondrial genome, their association with genes for ATPase subunits, and the hydrophobic nature of their putative translation products. It should be noted that some CMS-ORFs do not satisfy all of these criteria, and ORFs that satisfy these criteria are not necessarily associated with CMS. Therefore, it is difficult to infer the capability of MS induction of mitochondrial genomes solely from their nucleotide sequences. Morphological, physiological, and molecular biological studies suggest that multiple mechanisms cause CMS. Nuclear genes that suppress CMS have been identified. Post-transcriptional suppression of CMS-ORFs mediated by a certain class of RNA binding proteins (pentatrico peptide repeat proteins) is the predominant mechanism of fertility restoration. On the other hand, CMS suppression that is not associated with post-transcriptional suppression of CMS-ORFs has also been reported, suggesting that various types of gene-products are involved in fertility restoration

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

SUMMARYOf the two tRNA Cys (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA Cys2(GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA Cys2 (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA Cys2 (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA Cys1 (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.

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BackgroundObtaining 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.FindingsFive 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.ConclusionsEfficient 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s41065-016-0015-z) contains supplementary material, which is available to authorized users.

Cytoplasmic male sterility in plants is caused by male sterility-inducing mitochondria, which have emerged frequently during plant evolution. Nuclear Restorer-of-fertility (Rf) genes can suppress their cognate male sterility-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 homologues in plants. We analyzed 37 plant genomes and concluded that a single copy is the ancestral state in Caryophyllales. Among the sugar beet Oma1 homologues, 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 homologue 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 A. thaliana. This suggests a segmental duplication rather than a whole genome duplication as the mechanism of RF-Oma1 evolution. Among the positively selected codons in RF-Oma1, many 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 paralogue. 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.

Introns may be considered as optional because they are removed from mRNA molecules, but introns are fairly preserved for unknown reasons. Previously, the mitochondrial rps3 gene of sugar beet (Beta vulgaris L., Caryophyllales) was shown to represent a unique example of an intron loss. We have determined the distribution of the rps3 intron in 19 Caryophyllalean species. The intron was absent from the Amaranthaceae and the Achatocarpaceae. In the Caryophyllaceae, Dianthus japonicus rps3 was pseudogenized but the intronic sequence was retained. Intact intron-bearing rps3 copies were cloned from Portulaca grandiflora and Myrtillocactus geometrizans, members of the sister clade of the Amaranthaceae-Achatocarpaceae-Caryophyllaceae clade. Most of the C-to-U RNA-editing sites in Portulaca and Myrtillocactus rps3 transcripts were homologous in the two species as well as in the sugar beet rps3, which, unlike other 12 rps3 transcripts, lacks editing in the exonic regions around the intron. Provided that the loss of editing preceded the loss of rps3 intron, it appears conceivable that a requirement for editing could have prevented the loss of group-II introns retained in angiosperm mitochondrial genomes. This interpretation is an alternative to the conventional one that views the loss of editing as a mere trace of RNA-mediated gene conversion