A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Summary Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned -subunit of F₁-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.     

Reorganization of mitochondrial genomes of cytoplasmic revertants in cms-S inbred line WF9 in maize

Summary Cytoplasmic reversion to fertility in cms-S maize has been previously correlated with changes in mitochondrial genome organization, specifically with loss of the autonomously replicating linear plasmid-like DNAs, S1 and S2, and with accompanying alterations in the high molecular weight mtDNA (main genome) that specifically involved S1 and S2 sequences. These studies, however, dealt with cytoplasmic revertants occurring in the cms-VG M825 inbred line and in the cms-VG M825/Oh07 F₁ hybrid. This paper deals principally with patterns of mitochondrial DNA reorganization accompanying cytoplasmic reversion to fertility in the WF9 inbred line nuclear background. Here the free S1 and S2 plasmid-like DNAs are retained in the revertants. Mitochondrial DNA analysis by Southern hybridization using cloned fragments of S1 and S2 shows altered organization around S-homologous regions in the main mitochondrial genome of revertants as compared with that of the male-sterile parental controls, but the pattern of main genome changes involving these regions differs from that of the cytoplasmic revertants that occurred in M825 and M825/Oh07 backgrounds. Similar experiments using a clone of the cytochrome oxidase I (COX I) gene of maize as a probe indicate that reorganization in this region is also involved in the changes in mtDNA that accompany cytoplasmic reversion to male fertility in cms-S WF9. The heterogeneity in patterns of reorganization of the main mtDNA genome that accompany cytoplasmic reversion in the same and different nuclear backgrounds are discussed in relation to cytoplasmic male sterility (CMS).     

A comparative study of the mitochondrial genome organization in in vitro cultures of diploid, tetraploid, and hexaploid Triticum species

Southern-blot hybridizations of total DNA to mitochondrial DNA (mtDNA) probes were used to investigate the extent of mtDNA variability in cultures derived from immature embryos of diploid (Triticum monococcum, genomic formula: AA, T. tauschii, genomic formula: DD), allotetraploid (T. durum cv Creso, genomic formula: AABB), and allohexaploid (T. aestivum, genomic formula: AABBDD) wheat species. Similar distinct changes in mtDNA organization were observed in in vitro cultures of the derived tetraploid and the hexaploid species with related genomes. The tetraploid and hexaploid species share the B genome and mtDNA variability in in vitro culture is known to be under nuclear control. These results suggest that a study of B genome diploids and other polyploid combinations would now shed light on whether or not mtDNA variability in tissue cultures is under B-genome control.     

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid

Summary A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5 ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3 ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.     

RFLP Analysis for Mitochondrial Genome of CMS-Rice

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.     

细胞质雄性不育水稻线粒体基因组的RFLP分析

利用RFLP技术,比较研究了在农业生产上广泛应用的9种细胞质雄性不育体系的线粒体基因组,结果表明：1）9种水稻雄性不育细胞质的遗传相似性变化范围为0．615～1．000。所有配子体雄性不育细胞质的遗传相似性变化范围为0．6431．000,其中QXA,DY1A和YM15A这3种细胞质的遗传相似性为1．000。所有孢子体雄性不育细胞质的遗传相似性为1．000,2）在配子体细胞质雄性不育水稻中,用3个探针（atpa,atp9＋nad6,cox2）与2种内切酶（HindⅢ,BamHⅠ）的组合未发现不育系与保持系之间的多态性,但在探针atp6,cob,和had2的杂交带型中找到了不育系和保持系之间的一些差异。YTA和YTB在5个探针．内切酶组合（atp61HindⅢ,coblHindⅢ,atp61BamHⅠ,coblBamHⅠ,nad21BamHⅠ）中存在差异。3种细胞质（QX,SJ,DY1）的不育系和保持系之间的差异是相同的,都出现在atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ等3个组合中。YM15A和YM15B在4个组合（atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ,nad2／BamHⅠ）中存在差异。LYA和LYB的差异出现在cob／HindⅢ,cob／BamHⅠ,nad2／BamHⅠ这3个组合中;3）在孢子体细胞质雄性不育水稻中,所有不育系的带型是一样的,所有保持系的带型也一样。不育系和保持系的差异出现在atp6／HindⅢ,cob／HindⅢ,atp6／EcoR,Ⅰcob／EcoRⅠ,cox1／EcoRⅠ,atp6／BamHⅠ,cob／BamHⅠ,cox1／BamHⅠ,cox2／BamHⅠ等9个组合中。这些结果在分子水平上揭示了9种雄性不育细胞质的线粒体基因组存在结构多样性,并为其细胞质雄性不育分子机理的研究打下了基础。     

An S1 episomal gene of maize mitochondria is expressed in male sterile and fertile plants of the S-type cytoplasm

Summary Maize mitochondria of cytoplasmic male sterile (cms-S) plants contain two linear episomes, S1 (6397 bp) and S2 (5453 bp). S1 contains three long open reading frames URF2 (1017 bp), URF3 (2782 bp) and URF4 (768 bp). We have demonstrated that the URF3 sequence of S1 encodes a protein with an apparent molecular weight of 103 kDa which is found in cms-S but undetectable in cms-T, cms-C or normal (fertile) mitochondria. A translational fusion containing the 5 terminus of the lacZ gene and 800 bp of the 3 end of URF3 was isolated from a cms-S mitochondrial genomic library in the expression vector gt11. Polyclonal antibodies raised against the resulting fusion protein immunoprecipitated a 103 kDa polypeptide from among [³⁵S]-methionine-labeled cms-S mitochondrial proteins but not from normal mitochondrial proteins. The mitochondria of fertile F1 plants resulting from a cross between B37 cms-S and Ky21 (universal restorer) contain as much of this 103 kDa protein as is observed in sterile cms-S mitochondria. The mitochondria of fertile cytoplasmic revertants from cms-RD and cms-LM in a WF9 nuclear background also synthesized the 103 kDa protein. We conclude that the URF3 sequence of the S1 episome is expressed in vivo and that the presence of its gene product in maize mitochondria is not sufficient to confer the male sterile phenotype.     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

Mitochondrial genome organization and cytoplasmic male sterility in plants

Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes(rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterialrol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco andBrassica by genetic engineering methods.     

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension,callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the -subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.     

The Mitochondrial Genome Organization of a Maize Fertile Cmst Revertant Line Is Generated through Recombination between Two Sets of Repeats

The mitochondrial genome (mtDNA) organization from a fertile revertant line (V3) derived from the maize cytoplasmic male sterile type T (cmsT) callus tissue culture has been determined. We report that the sequence complexity can be mapped on to a circular ``master chromosome' of 705 kb which includes a duplication of 165 kb of DNA when compared to its male sterile progenitor. Associated with this event is also a 0.423-kb deletion, which removed the cmsT-associated urf13 gene. As found for the maize normal type (N) and cmsT mitochondrial genomes, the V3 master chromosome also exists as a multipartite structure generated by recombination through repeated sequences.     

Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS

Summary Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).     

Transfer of the Brassica tournefortii cytoplasm to B. napus for the production of cytoplasmic male sterile B. napus

The donor-recipient fusion method was used to combine the cytoplasm of Brassica lournefortii with the nucleus of B. napus for the production of cytoplasmic male sterile (CMS) plants. X-ray-irradiated mesophyll protoplasts of B. tournefortii were fused with iodoacetamide (lOA)-inactivated hypocotye protoplasts of B. napus . Selective conditions of IOA concentrations and X-ray doses were determined, which resulted in recovery of fusion products and inhibition of further growth of unfused parental cells. In total, 54 plants were obtained from different fusion experiments, of which 25 were verified as cybrids or partial hybrids. Mitochondrial DNA (mtDNA) analyses using 5 mitochondrial gene probes revealed that 20 of the 25 fusion-derived plants had mtDNA either identical, or with varying degrees of similarity, to B. lournefortii . These plants were classified into four groups on the basis of pollen viability and number. Seven plants were categorised as male sterile since they did not produce pollen or had non-viable pollen. Of the male sterile plants, five had a mtDNA pattern identical to B. tournefortii and a nuclear DNA content corresponding to B. napus . The nuclear-mito-chondrial constitution of these plants thus indicates that the combination of B. tournefortii cytoplasm with the B. napus nucleus results in CMS. Furthermore, mtDNA analysis of the two additional male sterile plants which displayed a rearranged mtDNA, revealed that the only mtDNA similarity shared among all male sterile plants was specific for B. tournefortii atp6 pattern. This indicates that the atp6 region of B. tournefortii may be involved in the expression of CMS.     

Characterization of a partial male fertility derived from Ogura-cms in progeny of a resynthesized Brassica napus

Summary The inheritance of a partial male fertile phenotype in somatic hybrid B. napus plants that carried novel mtDNA was investigated over five backcross generations to B. napus Triton. The recurrent parent and the original somatic hybrid both contained chloroplasts resistant to atrazine. The F₁ population contained mainly plants that were partial fertile, and some of the plants differed in mtDNA. The partial fertility predominated in the progeny of each backcross generation, but fully male sterile and fertile plants were also obtained. However, the sterility/fertility of these latter plants was not stable; both the fully male sterile and the male fertile plants produced progeny that were again predominantly partial male fertile. This pattern of predominant partial fertility but occasional sterile and fertile plants persisted in different nuclear backgrounds. Neither the male sterility nor the male fertility could be fixed and made stable. Test crosses indicated that restorer genes were probably not associated with appearance of male fertile plants. The evidence indicates that the behavior of the partial male fertility is cytoplasmic, and probably controlled by the chondriome.     

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.