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).     

Integrated R2 sequence in mitochondria of fertile B37N maize encodes and expresses a 130 kD polypeptide similar to that encoded by the S2 episome of S-type male sterile plants.

Expression of a 130 kDa protein from open reading frame 1 of the integrated form of the R2 mitochondrial plasmid in normal mitochondria of B37 and other inbred lines is described. The protein appears identical to that synthesized by the closely related S2 episome found in cytoplasmic male sterile maize of the S type. Protein was detected using antisera raised against a beta-galactosidase:ORF1 fusion product containing the most antigenic region of the ORF1 product. Detection of this protein is in contrast to previous reports that mitochondria of normal, male-fertile lines either do not contain this protein, or that there are 11 in-frame stop codons in the reading frame. The integrated R2 of B37N was cloned and this region sequenced, confirming that a continuous open reading frame existed. These results are discussed in relation to the possible role of the S-type episomes in causing cytoplasmic male sterility.     

Loss of CMS-specific mitochondrial DNA arrangement in fertile segregants of Petunia hybrids

The progeny of somatic hybrid Petunia plants derived from the fusion of a male-fertile line and a cytoplasmic male-sterile (cms) line were examined. Male-fertile progeny derived from three different male-sterile somatic hybrid plants did not exhibit the mitochondrial DNA (mtDNA) arrangement which has previously been correlated with cms in Petunia. The cms-associated mtDNA arrangement was present in the male-sterile predecessors of these fertile revertants. Thus, it is concluded that the loss of this mtDNA arrangement is associated with reversion to fertility in the progeny of the unstable somatic hybrid petunia plants.     

Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasms of maize (Zea mays L.)

Summary Differences in fertility restoration and mitochondrial nucleic acids permitted division of 25 accessions of S-type male sterile cytoplasm (cms-S) of maize into five subgroups: B/D, CA, LBN, ME, and S(USDA). S cytoplasm itself (USDA cytoplasm) was surprisingly not representative of cms-S, since only two other accessions, TC and I, matched its mitochondrial DNA pattern. CA was the predominant subgroup, containing 18 of the 25 accessions. The B/D and ME subgroups were the most fertile and LBN the most sterile. The exceptional sterility of LBN cytoplasm makes it the most promising of the 25 cms-S accessions for the production of hybrid seed. The most efficient means of quantifying the fertility of the subgroups was analysis of pollen morphology in plants having cms-S cytoplasm and simultaneously being heterozygous for nuclear restorer-of-fertility (Rf) genes. This method took advantage of the gametophytic nature of cms-S restoration. The inbred NY821LERf was found to contain at least two restorer genes for cms-S. Fertility differences were correlated with mitochondrial nucleic acid variation in the LBN, ME, and S (USDA) subgroups.Paper No. 9498 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Mutations leading to nuclear restoration of fertility in S male-sterile cytoplasm in maize

Summary Among the offspring of crosses involving S male-sterile shrunken-2 inbred lines and their corresponding isogenic maintainer lines a number of exceptional male-fertile plants were identified. Some of these were plants with entirely fertile tassels but most were chimeras involving both sterile and fertile tassel elements. The majority of male-fertile exceptional plants, upon crossing with male-sterile testers, produced male-sterile test-cross progeny, indicating that the male-fertile trait is not pollen transmissible. However, there were four separate instances, involving three of the inbred lines, in which the crosses with S male-sterile testers produced male-fertile progeny, indicating that the newly arisen male-fertile trait is pollen transmissible. In three of these cases, the male fertility can be traced to a single plant in essentially male-sterile families. The fourth evidently involved a change in a maintainer plant whose progeny thereafter segregated for the ability to restore S sterile cytoplasm. In all cases, the results of progeny tests are consistent with the gametophytic pattern of restoration associated with S male-sterile cytoplasm.The male-fertile exceptions described here can be accounted for formally as mutations at one or more restorer gene loci in the nucleus. Taking account of the fact that mutations of restorer genes have not been reported previously in maize, and that four such changes were encountered in the same strains in which we have identified other male-fertile exceptions involving change in the cytoplasm, we have suggested a common basis for the two kinds of events. According to this scheme, given the first appearance, by whatever process, of the male-fertile element in sterile cytoplasm, it may become established and continue to propagate either in the cytoplasm or in the nucleus. In the former case, the change registers as cytoplasmic and the new strain has the characteristics of a maintainer which transmits the male-fertile trait through the egg, but not the sperm; in the latter case, the change occurs in the nucleus and the new strain, now behaving as a restorer, transmits male fertility through both egg and sperm.Dedicated to Dr. M. M. Rhoades on the occasion of his 70th birthday.Research supported in part by CSRS grant 177-15-04, PL 89–106, and by NSF GB 29488.     

A protein specific to mitochondria from S-type male-sterile cytoplasm of maize is encoded by an episomal DNA

Mitochondria of S-type cytoplasmic male sterile maize contain two linear double-stranded DNA molecules, S1 and S2. Two open reading frames (ORF1 and ORF2) are present in S2 DNA. Fragments from ORF1 were inserted into plasmids to achieve expression in Escherichia coli. Cells transformed with recombinant plasmids produced mRNA which hybridized with ORF1 and corresponding polypeptides were synthesized by in vivo and in vitro systems. Antiserum against a lacZ/S2 fusion protein precipitated the anticipated polypeptides from transformed E. coli cells and was therefore used to detect homologous peptide sequences in protein preparations of mitochondria from different maize cytoplasms. The antiserum detected a protein of 125 000 M_r present in mitochondria from male sterile B73S but absent from the fertile B73N cytoplasm.     

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection

Summary Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.Scientific Journal Series Article no. 11,185 of the Minnesota Agricultural Experiment Station and no. 2295 of the Florida Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Cloning of mtDNA fragments homologous to mitochondrial S2 plasmid-like DNA in maize

Summary Mitochondria from S-type cytoplasmic male-sterile maize contain two small DNA species, S1 and S2, which are absent from other fertile and male-sterile cytoplasms. These species have been cloned in plasmid pBR322 by the homopolymer extension method. Probes made with these recombinant plasmids have been used to establish the homology between high molecular weight mitochondrial DNAs of fertile and male-sterile cytoplasms, and small mitochondrial plasmid-like molecules. Hybridization and mapping data show that S2 DNA copies are homologuous with sequences of the normal mitochondrial genome. A comparison of physical maps of different isolated mtDNA fragments indicates a heterogeneous arrangement of S2 sequences in the mtDNA population of normal fertile maize cytoplasm. The origin of this heterogeneity is discussed.     

Instability of s male-sterile cytoplasm in maize

A number of S male-sterile plants from several shrunken-2 inbred lines were crossed initially with an R138-TR inbred line pollinator carrying the nonrestoring genotype for S sterile cytoplasm. One such cross, involving a male-sterile female parent from inbred line M825, produced, unexpectedly, a number of male-fertile F₁ progeny, along with the expected male-sterile off-spring. Pollen records of plants in F₂, F₃ and F₄ progenies in the exceptional pedigree, and of a variety of testcross and backcross progenies from these male-fertile exceptions, indicate that the exceptional male fertility is not attributable to the action of either dominant or recessive nuclear restorer genes. They are, however, consistent with the hypothesis that the event responsible for the appearance of exceptional male-fertile offspring among progeny of the original cross involved a change from male-sterile to male-fertile condition in the cytoplasm of the male-sterile M825 plant involved as the female parent in this cross. It appears that this plant bore an ear in which there was a relatively early mutational event at the cytoplasmic level resulting in a chimera involving some kernels which carried S male-sterile cytoplasm, and others which carried the mutated fertile cytoplasmic condition. The finding of a number of additional ear chimeras supports this contention.—The evidence suggests that the change from sterile to fertile cytoplasm has occurred in a number of other instances. The male-sterile line M825 is especially prone to this change. These findings are of particular interest because it has heretofore been considered that both S and T types of male-sterile cytoplasm are highly stable.—The data presented here are not sufficient to support the notion that the exceptional event involves a qualitative change, analogous to gene mutation, in a cytoplasmic entity governing the expression of male fertility. It is equally plausible that the exceptional male fertility is the result of occasional transfer of normal cytoplasm through the male germ cells of maintainer parents.     

Fertile revertants from S-type male-sterile maize grown in vitro

Summary Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3–4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13–20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.     

Molecular analysis of the inheritance and stability of the mitochondrial genome of an inbred line of maize

Summary We have investigated the inheritance of the mitochondrial DNA (mtDNA) restriction endonuclease digestion patterns of maize inbred line B37N in individual plants and pooled siblings in lineages derived from five separate plants in the third generation following successive self-pollinations. The restriction fragment patterns of the different mtDNA samples were compared after digestion with five endonucleases. No differences were visible in the mobilities of the 199 fragments scored per sample. Hybridization analysis with two different cloned mtDNA probes, one of which contains homologies to a portion of the S2 plasmid characteristic of cms-S maize, failed to reveal cryptic variation. The apparent rate of genomic change in maize mtDNA from inbred plants appears to be very slow, compared with the faster rates of change seen in maize tissue cultures and with the documented rapid rate of inter- and intraspecific variation for mammalian mtDNA.     

Classification of normal and male-sterile cytoplasms in maize. I. Electrophoretic analysis of variation in mitochondrially synthesized proteins

Male-sterile cytoplasms of maize have previously been classified into three groups (T, S and C) according to their fertility ratings in various inbred backgrounds. In earlier studies, mitochondria from three male-sterile cytoplasms, representing each of these three groups, have been found to synthesize characteristic variant polypeptides that distinguish them from each other and from those of normal (N) cytoplasm. In order to determine the extent of cytoplasmic variation, we have now analyzed the translation products of mitochondria from 28 additional cytoplasmic sources. The results show that on this basis 18 of the cytoplasms are identical to the USDA (S) cytoplasm, three are identical to the Texas (T) cytoplasm and two are identical to the C cytoplasm. The five remaining cytoplasms are indistinguishable from normal, male-fertile (N) cytoplasm. Our classification of the cytoplasms is in general agreement with those based on fertility restoration. However, of three cytoplasms that have previously remained unclassified, two (B and D) have now been assigned to the S group and one (LF) to the N group. No heterogeneity in mitochondrial translation products was detected within the normal or any of the three male-sterile groups. The usefulness of the analysis of mitochondrial translation products as a method for classifying normal and male-sterile cytoplasms is discussed.     

Polymorphism of mitochondrial DNA ‘S’ regions among normal cytoplasms of maize

Genomic variation in S1 and S2 homologous sequences, defined as the S regions, were examined in mitochondrial DNAs of 12 normal cytoplasm maize lines collected in the United States. Three genomic variants were detected among the 12 cytoplasms, eight of which were identical to the Wf9 model structure. Hybridization data with S1 and S2 DNAs and with two cosmids spanning these regions were consistent with the concept that S1 and S2 sequences are found in each normal cytoplasm. Three variations of the S1 region were established; the Wf9 structure, a second group consisting of F6, A188, and W182BN, and a third, Black Mexican. Genome structure was conserved through the S2 region in all lines examined. None of the cytoplasms included complete copies of S1; the 1400 bp repeat characteristic of S1 and S2 was absent in the S1 region of all lines. A 2.1 kb linear DNA was observed instead of a 2.3 kb DNA in F6, A188, and W182BN. Integrated copies of S1 and S2 sequences may be a constituitive characteristic of normal, male-fertile maize cytoplasms.     

The linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase

Summary Plasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced. The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs. The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases. The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages. ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively. No homology to any published protein sequence was found for the smaller ORFs. The origin of the terminal protein attached to the 5 ends of pClK1 remains open; several alternatives for its origin are discussed. The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties. Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.     

Analysis of N-acetoxy-N-2-acetylaminofluorene-induced frame-shifts in pBR322

We present a simple system which can be used to study directly directly the sequence change and the cellular repair functions involved in frame-shift mutagenesis by a covalently reactive mutagen. Positive (+S) and negative (?S) alterations in the number of base pairs of the Tc gene of pBR322 were generated and particular clones with Ap^RTc^S phenotypes were selected for mutagenesis experiments. Exposure of these frame-shifted plasmid DNAs to a potent carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), in vitro, caused covalent alterations to DNA sequence and resulted in a number of revertants (Ap^RTc^R) not observed in the untreated controls. The dose curve indicated an exponential response suggesting single-hit kinetics. Differential inactivation of the Ap gene was observed among various E. coli strains. The wild-type AB1157 and AB2463 (yrecA) showed a similar dose curve while AB1886 (uvrA) showed a marked decrease in Ap^R clones at the same dose. Both addition (+S) and deletion (?S) plasmids exhibited similar dose curves on inactivation of Ap gene. The reversion frequency, however, of ?S plasmid was a factor of 10 times higher than +S plasmid. The reversion frequency also increase markedly with uvrA host but not with recA host. 2 types of deletion revertants of the +S plasmid were found. 1 revertant has a single GC base-pair deletion in GC-rich region which is likely to be a target for AAAF reaction. The other showed a deletion of 4 base pairs (TCGA) at the tandem repeating sequence TCGATCGA which may represent a hot spot for frame-shift mutation.     

Nuclear-cytoplasmic male-sterility in diploid dandelions

Male-sterility was found in diploid dandelions from two widely separated populations from France, and its inheritance was analysed by crossing a diploid male-sterile dandelion to diploid sexuals and triploid apomicts. Nuclear genetic variation, found in full-sib families, segregated for male-fertility, partial male-sterility, and full male-sterility, and also segregated for small-sized versus normally sized pollen. The crossing results are best explained by a cytoplasmic male-sterility factor in combination with two dominant restorer genes. Involvement of the cytoplasmic male-sterility factor was further investigated by chloroplast haplotyping. Male-sterility was exclusively associated with a rare chloroplast haplotype (designated 16b). This haplotype was found in seven male-sterile plants and one (apparently restored) male-fertile individual but does not occur in 110 co-existing male-fertile plants and not in several hundreds of individuals previously haplotyped. Apomicts with cytoplasmic male sterility were generated in some test crosses. This raises the question as to whether the male sterility found in natural dandelion apomicts, is of cytoplasmic or of nuclear genetic nature. As many breeding systems in Taraxacum are involved in shaping population structure, it will be difficult to predict the evolutionary consequences of nuclear-cytoplasmic male-sterility for this species complex.     

Variation in pollen production and pollen viability in natural populations of gynodioecious Beta vulgaris ssp. maritima: evidence for a cost of restoration of male function?

Gynodioecious species are defined by the co-occurrence of two clearly separated categories of plants: females and hermaphrodites. The hermaphroditic category may, however, not be homogeneous, as male fitness may vary among hermaphrodites as a result of many biological factors. In this study, we analysed estimates of pollen quantity and viability in the gynodioecious Beta vulgaris ssp. maritima, comparing hermaphrodites bearing a male-fertile cytotype and hermaphrodites bearing cytoplasmic male sterility (CMS) genes, which are counteracted by nuclear restoration factors. We show that: (i) pollen quantity continuously varies among restored hermaphrodites, suggesting a complex genetic determination of nuclear restoration; (ii) pollen viability was lower in restored (CMS) hermaphrodites than in non-CMS hermaphrodites, probably because of incomplete restoration in some of these plants; and (iii) pollen quantity and viability also varied among hermaphrodites with male-fertile cytotypes, possibly a result of a silent cost of restoration. Finally, we discuss the consequences of these results for pollen flow and the dynamics of gynodioecy.