High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis

• Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology.
• We used knock‐down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana.
• Knock‐down of RPS9M in the rps9m‐3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m‐3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m‐3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS.
• Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

     

The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats

A single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. A map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687-amino-acid protein with a predicted mitochondrial targeting pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis, although a smaller and related PPR gene is found about 40 kb from this site. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.     

Stable native RIP9 complexes associate with C‐to‐U RNA editing activity,PPRs, RIPs,OZ1, ORRM1 and ISE2

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C‐to‐U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co‐immunoprecipitation (co‐IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size‐exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high‐MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low‐MW inactive fractions. Active editing factor complexes were affinity‐purified using anti‐RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co‐IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C‐to‐U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.     

Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear‐encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)‐type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD–CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS‐associated gene in LD–CMS rice, similar to its role in BT–CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT–CMS rice. We also show that RF2 promotes degradation of atp6–orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT–CMS rice. The amount of ORF79 protein in LD–CMS rice was one‐twentieth of the amount in BT–CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD–CMS and BT–CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD–CMS and BT–CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD–CMS and BT–CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.     

The petunia restorer of fertility protein is part of a large mitochondrial complex that interacts with transcripts of the CMS-associated locus

A class of nuclear genes termed "restorers of fertility" (Rf) acts to suppress the expression of abnormal mitochondrial genes associated with cytoplasmic male sterility (CMS). In petunia, both the nuclear Rf gene and mitochondrial CMS-associated gene have previously been identified. The CMS-associated gene is an aberrant chimera in which portions of several mitochondrially encoded genes are fused to an unknown reading frame. The dominant Rf allele reduces the CMS-associated protein to nearly undetectable levels and alters the RNA population derived from the CMS locus, but its mechanism of action has not been determined. The petuniaRf gene is a member of the pentatricopeptide repeat gene family (PPR), an unusually large gene family in Arabidopsis (approximately 450 genes) compared with yeast (five genes) and mammalian genomes (six genes). The PPR gene family has been implicated in the control of organelle gene expression. To gain insight into the mode of action of PPR genes, we generated transgenic petunia plants expressing a functional tagged version of Rf. Analysis of the restorer protein revealed that it is part of a soluble mitochondrial inner-membrane-associated, RNase-sensitive high-molecular-weight protein complex. The complex is associated with mRNA derived from the CMS locus.     

The Arabidopsis mitochondrial membrane‐bound ubiquitin protease UBP27 contributes to mitochondrial morphogenesis

Mitochondria are essential organelles with dynamic morphology and function. Post‐translational modifications (PTMs), which include protein ubiquitination, are critically involved in animal and yeast mitochondrial dynamics. How PTMs contribute to plant mitochondrial dynamics is just beginning to be elucidated, and mitochondrial enzymes involved in ubiquitination have not been reported from plants. In this study, we identified an Arabidopsis mitochondrial localized ubiquitin protease, UBP27, through a screen that combined bioinformatics and fluorescent fusion protein targeting analysis. We characterized UBP27 with respect to its membrane topology and enzymatic activities, and analysed the mitochondrial morphological changes in UBP27T‐DNA insertion mutants and overexpression lines. We have shown that UBP27 is embedded in the mitochondrial outer membrane with an N_in–C_out orientation and possesses ubiquitin protease activities in vitro. UBP27 demonstrates similar sub‐cellular localization, domain structure, membrane topology and enzymatic activities with two mitochondrial deubiquitinases, yeast ScUBP16 and human HsUSP30, which indicated that these proteins are functional orthologues in eukaryotes. Although loss‐of‐function mutants of UBP27 do not show obvious phenotypes in plant growth and mitochondrial morphology, UBP27 overexpression can change mitochondrial morphology from rod to spherical shape and reduce the mitochondrial association of dynamin‐related protein 3 (DRP3) proteins, large GTPases that serve as the main mitochondrial fission factors. Thus, our study has uncovered a plant ubiquitin protease that plays a role in mitochondrial morphogenesis possibly through modulation of the function of organelle division proteins.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica

The plant‐specific pentatricopeptide repeat (PPR) proteins with variable PPR repeat lengths (PLS‐type) and protein extensions up to the carboxyterminal DYW domain have received attention as specific recognition factors for the C‐to‐U type of RNA editing events in plant organelles. Here, we report a DYW‐protein knockout in the model plant Physcomitrella patens specifically affecting mitochondrial RNA editing positions cox1eU755SL and rps14eU137SL. Assignment of DYW proteins and RNA editing sites might best be corroborated by data from a taxon with a slightly different, yet similarly manageable low number of editing sites and DYW proteins. To this end we investigated the mitochondrial editing status of the related funariid moss Funaria hygrometrica. We find that: (i) Funaria lacks three mitochondrial RNA editing positions present in Physcomitrella, (ii) that F. hygrometrica cDNA sequence data identify nine DYW proteins as clear orthologues of their P. patens counterparts, and (iii) that the ‘missing’ 10th DYW protein in F. hygrometrica is responsible for two mitochondrial editing sites in P. patens lacking in F. hygrometrica (nad3eU230SL, nad4eU272SL). Interestingly, the third site of RNA editing missing in F. hygrometrica (rps14eU137SL) is addressed by the DYW protein characterized here and the presence of its orthologue in F. hygrometrica is explained through its simultaneous action on site cox1eU755SL conserved in both mosses.