A cytoplasmic male sterility-associated mitochondrial peptide in common bean is post-translationally regulated.

Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-or f 239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome. The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally.     

三例小麦细胞质雄性不育系线粒体DNA的扩增片段长度多态性分析

细胞质雄性不育是小麦杂种优势利用的重要途径,为了鉴定3例小麦雄性不育系的细胞质类型,对其线粒体DNA(mtDNA)进行扩增片段长度多态性(Amplified fragment length polymorphism,AFLP)分析。文中利用差速离心法和不连续蔗糖密度梯度超速离心法提取纯化小麦线粒体。结果表明:通过该提取方法获得的mtDNA,其质量和纯度能够满足PCR反应和遗传学分析。在64对选扩引物中,筛选到了4对特异性引物,其中引物E1/M7在ms(Kots)-90-110不育系扩增出3条特异条带;引物E4/M2在ms(Ven)-90-110不育系扩增出2条特异条带;引物E7/M6在ms(S)-90-110不育系中扩增出2条特异条带;引物E6/M4在ms(Kots)-90-110不育系中扩增出2条特异条带。这些特异引物可以用来作为鉴定具有粘果山羊草Aegilops kotschyi、偏凸山羊草Ae.ventricosa、斯卑尔脱小麦Triticum spelta 3类不育细胞质型小麦雄性不育系的细胞质分子标记,为研究小麦细胞质雄性不育机理奠定了分子基础。     

A mutation causing variegation and abnormal development in tobacco is associated with an altered mitochondrial DNA

A variegated mutation appeared in the leaves of a tobacco cybrid plant resulting from fusion of protoplasts from tobacco with Petunia . The mutation was inherited maternally. The light green coloration of leaf sectors resulted from a substitution of spongy parenchyma for palisade parenchyma. No defects were detected in the chloroplasts of the plants, which were derived from Petunia . The mitochondria, as judged by the electrophoretic pattern of their DNA after digestion with restriction endonucleases, were very similar to mitochondria of tobacco, although with some unique cybrid-specific fragments. A second round of fusions was performed to confirm that mitochondria, rather than chloroplasts, were associated with the variegated phenotype. In these fusions, the Petunia chloroplasts of the variegated plants were replaced by tobacco chloroplasts. The mitochondria, according to the DNA restriction pattern, retained all or some of the unique cybrid-specific fragments found in the original variegated tobacco cybrid. Since the variegated phenotype remained after the chloroplast exchange, the chloroplast DNA cannot be the site of the mutation which is responsible for the mutant phenotype. This result eliminates the chloroplast and confirms that the mitochondrial genome is associated with the mutant phenotype.     

Studies on wheat mitochondrial DNA organization. Comparison of mitochondrial DNA from normal and cytoplasmic male sterile varieties of wheat

The mitochondrial genome of fertile, male-sterile and restored cytoplasm lines of wheat has been studied by means of recombinant DNA and hybridization techniques. Using cloned fragments of mitochondrial DNA (mtDNA) from fertile wheat cytoplasms as probes, about 40% of the genome is shown to have a differential hybridization pattern. The use of wheat rRNA and corn cytochrome oxidase subunit II probes indicates that duplication and rearrangement of genes or parts of genes may account for the differences observed. DNA synthesis in isolated mitochondria showed neither preferential labeling of part of the mtDNA nor the presence of extrachromosomal elements.     

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.     

Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.     

Chloroplast DNA polymorphism in different types of cytoplasmic male sterile rice

The lengths of open reading frame (ORF)100 and ORF29-TrnC^GCA, the intronic sequence of rps16 and the transcribed spacer of TrnT^UGU-TrnL^UAA in chloroplast from different lines of cytoplasmic male sterility (CMS) rice were studied using indica types, japonica types and common wild rice as controls. The results show that the lengths of ORF100 and ORF29-TrnC^GCA in CMS lines are similar to those of typical indica. The sequences of the rps16 intron and the TrnT^UGU-TrnL^UAA spacer in sporophyte sterile types (wild-abortive type, Yinshui type and K type) are almost the same, and they also share a molecular marker of GTTGAG at nucleotide positions 220–225 in the rps16 intron. Therefore, it is speculated that the source of these three types is the same. In contrast, a gametophyte sterile type, Yuetai A does not contain such a GTTGAG sequence in the rps16 intron and has a unique G at position 595, which may works as a molecular marker distinguishing the sporophyte sterile type from the gametophyte sterile type. Based on the observation that CMS rice has much lower cytoplasmic polymorphism than indica, japonica and wild rice, it is concluded that CMS rice lack cytoplasm diversity. Therefore, it is important to introduce new sources of cytoplasm into hybrid rice.     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

Mitochondrial DNA modifications associated with cytoplasmic male sterility in rice

Summary Mitochondrial DNA was isolated from fertile and cytoplasmic male sterile lines of rice. Restriction analysis showed specific modifications in the male sterile cytoplasm. In addition to the major mitochondrial DNA, three small plasmid-like DNA molecules were detected by agarose gel electrophoresis in both cytoplasms. An additional molecule was specifically found in the sterile cytoplasm. These mitochondrial DNA modifications support the hypothesis of the mitochondrial inheritance of the cytoplasmic male sterility in rice.     

The expression of alternative oxidase and alternative respiratory capacity in cytoplasmic male sterile common bean

Summary The alternative respiratory pathway is present in all plant species investigated to date. Yet, the role of the alternative pathway is not clear. Some evidence suggests an important role in pollen development. We undertook this study to investigate the expression of alternative oxidase, in comparison with expression of a component of cytochrome oxidase, during pollen formation in common bean (Phaseolus vulgaris L.). Expression was evaluated immunohistochemically. In addition, we compared both the alternative oxidase capacity in young seedling tissues and alternative oxidase expression in developing flower buds of isonuclear cytoplasmic male sterile and male fertile bean lines. We observed no evidence of an association between the abnormal pollen development of CMS bean and changes in alternative oxidase expression or capacity. We did observe a tissueand stage-specific pattern of expression of alternative oxidase, differing from the expression pattern of cytochrome oxidase subunit II, during anther development in normal bean lines. Although no association was evident between the cytoplasmic male sterility phenotype and differential expression of alternative oxidase, the regulated pattern of alternative oxidase expression in developing anthers does suggest that the alternative pathway may play a role in microgametogenesis and microsporogenesis.