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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