Françoise Budar

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.

Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica cybrids.

SummarySequence analysis of the Ogura-specific mitochondria) DNA (mtDNA) fragment isolated previously from Brassica cybrids carrying Ogura cytoplasmic male sterility (cms) revealed a tRNAfMet sequence, a putative 138 amino acid open reading frame (orf138), and a 158 amino acid ORF (orf158) previously observed in mitochondria) genomes from several other plant species. Transcription mapping showed that both ORFs are present on a 1.4 kb cms-specific transcript. The orf158 sequence is also transcribed in fertile plants on a different mRNA, and thus is unlikely to be related to cms. On the other hand, fertile revertant plants lack transcripts of the orf138 sequence, whose possible role in the mechanism of Ogura cms is discussed.

Male sterility in plants: occurrence, determinism, significance and use.

Most of higher plant species are hermaphroditic and male-sterility is often considered as an accident of development. In fact among the multiple possible causes of male-sterility, the most frequently met in nature is cytoplasmic male-sterility (cms) which is a maternally inherited trait playing an active role in the evolution of gynodioecious species. Recent molecular studies have shown that this trait is determined by additional genes created in plant mitochondrial genomes due to their high recombinogenic activity. The physiological mechanisms by which the products of these genes interfere with the formation of male gametophytes are still the subject of intense research.

Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids

Transcription of a putative mitochondrial gene (orf138) has previously been correlated with Ogura cytoplasmic male-sterility (CMS) in rapeseed cybrids. In this paper, studies performed on a Brassica cybrid with a different organization of the orf138 locus confirm this association. We also show that mitochondria isolated from male-sterile rapeseed plants synthesize a polypeptide of 19 kDa, which is absent in fertile revertants. Antibodies against a glutathione S-transferase-ORF138 fusion protein were raised to establish that this 19 kDa polypeptide is the product of orf138. The anti-ORF138 serum was used to demonstrate that the orf138 translation product occurs only in sterile cybrids and co-purifies with the mitochondrial membrane fraction.

A 2.5 kb NcoI fragment of Ogura radish mitochondrial DNA is correlated with cytoplasmic male-sterility in Brassica cybrids

SummarySpontaneous reversion to fertility was studied in the progeny of a cytoplasmic male-sterile (CMS) Brassica napus cybrid containing recombinant B. napus/Ogura radish mitochondrial genomes. This reversion is concomitant with the disappearance of a 2.5 kb NcoI fragment present in the mitochondrial DNA of Ogura radish, and of CMS cybrids derived from plants carrying Ogura cytoplasm, and absent in the mitochondrial genome of normal Brassicas and fertile cybrids. This specific fragment hybridizes to a 1.4 kb transcript found only in male-sterile plants bearing an Ogura derived cytoplasm.

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.

Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids

Abstract A PCR analysis of mitochondrial (mt) genomes of cybrid rapeseed plants revealed substoichiometric concentrations of molecules bearing different configurations of the gene (orf138) responsible for Ogura cytoplasmic male sterility (CMS). These sub-stoichiometric molecules are also present in plants bearing the unmodified Ogura cytoplasm. In one cybrid family, which shows reversion of the male sterile phenotype, we observed changes in the respective proportions of these molecules. The phenotypic (sterility-fertility) reversion occurs as a result of a modification of the equilibrium state between the different forms of the orf138 gene and is very probably determined by the level of expression of this gene. Stable situations are always characterized by one predominant form; the others, when present, exist in substoichiometric amounts. We report results indicating that the different forms of the orf138 gene are continuously interconverted by recombination and that an active mechanism is involved in the maintenance of some substoichiometric molecules.

The steady‐state level of mRNA from the Ogura cytoplasmic male sterility locus in Brassica cybrids is determined post‐transcriptionally by its 3′ region

We have investigated the control of the expression of three different configurations of the mitochondrial gene orf138, whose expression is correlated with Ogura cytoplasmic male‐sterility in rapeseed cybrids. These configurations, termed Nco2.5/13S, Nco2.7/13F and Bam4.8/18S, specific to the 13S (sterile), 13F (fertile) and 18S (sterile) cybrids respectively, have the same 5′ regions but different 3′ regions. The orf138 transcript from Bam4.8/18S is 10‐fold more abundant than the one from Nco2.5/13S, while no orf138 transcript from Nco2.7/13F accumulates. However, transcriptional activity measurements show that the rate of transcription is equivalent for the three configurations. These results strongly suggest that the steady‐state level of mRNA from the orf138 locus is determined post‐transcriptionally, most likely by its 3′ region. To determine the role of these 3′ regions, we have established an in vitro decay and processing system. In the presence of rapeseed mitochondrial lysate, synthetic RNAs corresponding to the 3′ region of the Nco2.7/13F transcript are, as expected, less stable than RNAs corresponding to the 3′ regions of the Nco2.5/13S and Bam4.8/18S transcripts. We have also observed in vitro processing of synthetic RNAs at the sites corresponding to the 3′ ends of the natural mRNAs from Nco2.5/13S and Bam4.8/18S. Further analysis of the role of these 3′ regions in in vitro RNA stability should help us to better understand post‐transcriptional control in plant mitochondria.

The restorer Rfo gene acts post-translationally on the stability of the ORF138 Ogura CMS-associated protein in reproductive tissues of rapeseed cybrids

This paper describes the analysis of the effect of the restorer gene Rfo on the expression of the ORF138 protein associated with Ogura cytoplasmic male sterility (CMS) which has been engineered in rapeseed by protoplast fusion. We show that the presence of the Rfo gene in the genome of the plants decreases the amount of ORF138 protein in floral buds, this effect being the most dramatic in anthers at the stage of development when the sterile phenotype is normally expressed. However, the amount of orf138 transcripts is not affected by the Rfo gene in the same organs at the same stages. Total polysome analyses of buds and anthers show that the orf138 transcripts are translated with the same efficiency in sterile and restored plants. From these results we infer that the Rfo gene product acts on the post-translational stability of the ORF138 protein, leading to a decrease in the accumulation of the protein and a restoration of fertility.