A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129 ) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST–ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.     

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.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

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.     

甘蓝型油菜线粒体功能未知基因 ORF 117的载体构建与转化

本研究克隆了甘蓝型油菜线粒体功能未知基因 ORF 117,构建了带有线粒体转运信号肽序列（TP ）的植物过表达载体35S ∷TP-ORF117、35S ∷TP-EGFP 和35S ∷TP-ORF117-EGFP ,转化野生型拟南芥并进行抗除草剂筛选,共获得纯合的转基因拟南芥株系62个。qPCR 表明,ORF 117在转基因材料中得到高效过表达。用转35S ∷TP-EGFP 、35S ∷TP-ORF117-EGFP 拟南芥制备原生质体,经线粒体特异染料染色,在激光共聚焦显微镜下做线粒体、GFP 共定位检测,GFP 蛋白和 ORF117-EGFP 融合蛋白被准确定位到了线粒体。     

The 5′-leader sequence of sugar beet mitochondrial <Emphasis Type="Italic">atp6</Emphasis> encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a mitochondrially encoded trait, which is characterized by a failure of plants to produce viable pollen. We have investigated the protein profile of mitochondria from sugar beet plants with normal (fertile) or CMS cytoplasm, and observed that a 35-kDa polypeptide is expressed in Owen CMS plants but not in normal plants. The variant 35-kDa polypeptide was found in CMS mitochondria placed in five different nuclear backgrounds. Interestingly, this polypeptide proved to be antigenically related to a 387-codon ORF (preSatp6) that is fused in-frame with the downstream atp6. The presequence extension of the atp6 ORF is commonly found in higher plants, but whether or not it is normally expressed has hitherto remained unclear. Our study is thus the first to demonstrate that the atp6 presequence is actually translated in mitochondria. We also observed that preSATP6 is a mitochondrial membrane protein that assembles into a homogeneous 200-kDa protein complex. In organello translation experiments in the presence of protease inhibitors showed a reduction in the abundance of mature preSATP6 with time, suggesting that the mature preSATP6 may be derived by proteolytic processing of a translation product of the preSatp6/Satp6 ORF.     

Mitochondrially-targeted expression of a cytoplasmic male sterility-associated <Emphasis Type="Italic">orf220</Emphasis> gene causes male sterility in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Background  

The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.     

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.