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.     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

Genetic characterization of a pentatricopeptide repeat protein gene,orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.     

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.     

Molecular markers linked to the Rf2 fertility restorer gene in cotton.

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.     

Genetic analysis of fertility restoration and accumulation of ORF125 mitochondrial protein in the kosena radish (Raphanus sativus cv. Kosena) and a Brassica napus restorer line

The genetics of fertility restoration (Rf) of kosena radish CMS has been characterized. The kosena CMS-Rf system is genetically the same as that of the ogura CMS-Rf system. Two dominant genes that act complementary to the restoration of fertility control fertility restoration in kosena CMS. One allele (Rf1) is associated with accumulation of the CMS-associated protein, ORF125. The interaction of Rf1 and another allele (Rf2) was essential for the restoration of fertility in radish, whereas Rf1 alone was sufficient for the complete restoration of fertility in the B. napus kosena CMS cybrid. Received: 13 August 1999 / Accepted: 16 September 1999     

The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein

Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.     

A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice

A fertility restorer gene (Rf-1) of [ms-bo] cytoplasmic male sterility (CMS) in rice has been reported to be responsible for the processing of RNA of aberrant atp6 of mitochondria. We have carried out map-based cloning of the Rf-1 gene and found that a 4.7-kb genomic fragment of a restorer line promoted the processing of aberrant atp6 RNA when introduced into a CMS line. The genomic fragment contained a single open reading frame encoding 18 repeats of the 35 amino acid pentatricopeptide repeat (PPR) motif. The cloned PPR gene is a possible candidate of Rf-1. A non-restoring genotype was identified to have deletions within the coding region.     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

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.     

Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice

Commercial exploitation of heterosis is essential for enhancing productivity of rice. The use of cytoplasmic male sterility (CMS) and fertility restoration system greatly facilitates large scale production of hybrid seed. The wild abortive (WA) cytoplasm is most widely used for hybrid seed production in rice. The present study was undertaken to develop molecular markers for both WA cytoplasm based male sterility and its fertility restoration for use in efficient hybrid breeding. High degree of genetic differentiation of WA-cytoplasm from its normal fertile counterpart was observed due to DNA rearrangements involving five (coxI, coxIII, cob, atp6 and rps3) mitochondrial genes. Cleaved amplified polymorphic sequence (CAPS) markers based on five mitochondrial genes namely, coxIII, cob, atp9, rps3 and 18SrRNA polymorphic between CMS and maintainer line were developed. The utility of these informative markers was demonstrated in purity testing of the CMS line Pusa6A being used in commercial hybrid seed production. Fertility restoration was found to be controlled by a major locus in the Basmati restorer line PRR78, which was mapped to a short marker interval of 0.8 cM and a physical interval of 163.6 kb on rice chromosome 10. A total of 13 pentatricopeptide repeat (PPR) motif containing genes were predicted in a 1.66 Mb region on the long-arm of this chromosome of which, four were present in the marker interval containing the fertility restorer gene. High degree of conservation of gene order was observed between japonica and indica for the predicted PPR genes. A sequence tagged site (STS) and a genic non-coding microsatellite (GNMS) marker were designed based on one of the candidate PPR motif containing genes present in the marker interval, which were validated using F₂ population and other known restorer lines. The candidate gene based marker identified in the present study would be useful in marker assisted selection (MAS) for fertility restorer gene in hybrid breeding programme based on WA-CMS of rice.     

CCACA碱基序列是玉米CMS-S线粒体orf355-orf77转录本剪切识别位点

玉米S型细胞质雄性不育系（CMS-S）及其近等基因恢复系是研究核 质互作机制的重要遗传资源和理想模式体系．目前认为,CMS-S花粉败育是由其线粒体内细胞质不育基因orf355-orf77表达的毒性蛋白引起,而核育性恢复基因Rf3可通过引发orf355-orf77转录本的降解而解除其毒性作用,使花粉育性得以恢复．本研究采用Northern杂交和3′-RACE技术确定了orf355-orf77转录本的剪切位点,并发现在育性恢复的花粉中,orf355-orf77转录本被剪切成小片段之后聚合了poly(A)序列,推测这一过程加速了mRNA分子的降解,是育性恢复的关键环节．利用生物信息学方法分析了orf355-orf77转录本6个剪切位点的侧翼序列,发现在剪切位点下游10个碱基的位置均含有5′-CCACA-3′序列,推测该序列受到特定功能蛋白的识别,然后募集核酸内切酶对其进行剪切．研究结果可为揭示玉米CMS S育性恢复机理提供重要的理论依据．